JP2023047344A - Method for producing artificial three-dimensional tissue, method for measuring artificial three-dimensional tissue, and artificial three-dimensional tissue - Google Patents

Abstract

To provide methods for producing an artificial three-dimensional tissue that facilitates the observation of sensory nerve cells.SOLUTION: The method of the present invention comprises: preparing a culture vessel having a culture space surrounded by side walls and anchor portions provided on the opposing side walls; culturing in a culture tank to obtain a gel body having a first culture body in which dermal cells are cultured and a second culture body in which spheroids densely packed with sensory nerve cells are cultured; and culturing epidermal cells arranged on the gel body.SELECTED DRAWING: Figure 8

Description

TECHNICAL FIELD The present invention relates to an artificial three-dimensional tissue manufacturing method, an artificial three-dimensional tissue measuring method, and an artificial three-dimensional tissue.

Sensory nerves pass through human skin and receive various sensations. Various studies have been conducted on skin sensation, but many points remain unexplained. Therefore, a method of culturing sensory nerve cells inside a skin model has been carried out.

For example, Non-Patent Document 1 discloses that skin tissue and sensory nerve cells are cultured using a co-culture system using sponge collagen to prepare a wound healing model.

M. Blais, L. Mottier, M. A. Germain, S. Bellenfant, S. Cadau, and F. Berthod, “Sensory neurons accelerate skin reepithelialization via substance P in an innervated tissue-engineered wound healing model,” Tissue Engineering - Part A, vol.20, no.15-16, pp.2180-2188, 2014.

The method disclosed in Non-Patent Document 1 has a problem that it is difficult to observe and perform functional analysis because sensory nerve cells are scattered in the skin tissue.

The present invention has been made in consideration of the above points, and aims to provide a method for producing an artificial three-dimensional tissue and an artificial three-dimensional tissue that facilitate observation of sensory nerve cells.

Another object of the present invention is to provide a method for measuring an artificial three-dimensional tissue that facilitates functional analysis of sensory nerve cells.

A first aspect of the present invention is to prepare a culture tank having a culture space surrounded by side walls and anchor portions provided on the opposing side walls, and perform culture in the culture tank to obtain dermis. obtaining a gel body having a first culture body in which cells are cultured and a second culture body in which spheroids densely packed with sensory nerve cells are cultured; and culturing epidermal cells arranged on the gel body. A method for producing an artificial three-dimensional tissue, comprising:

A second aspect of the present invention is to prepare a measuring instrument in which a plurality of electrodes are arranged in an array and measure an extracellular potential with the electrodes, and the artificial three-dimensional tissue of the first aspect of the present invention. and placing the artificial three-dimensional tissue manufactured by the manufacturing method on the electrode.

A third aspect of the present invention is a dermal tissue layer containing dermal cells, an epidermal tissue layer containing epidermal cells and formed on the dermal tissue layer, contained in the dermal tissue layer, from the dermal tissue layer to the and spheroids densely packed with sensory nerve cells having neurites extending toward the epidermal tissue layer.

The present invention can provide a method for producing an artificial three-dimensional tissue and an artificial three-dimensional tissue that facilitate observation of sensory nerve cells.

In addition, the present invention can provide a method for measuring an artificial three-dimensional tissue that facilitates functional analysis of sensory nerve cells.

1 is a perspective cross-sectional view schematically showing an artificial skin tissue 1 according to an embodiment of the present invention; FIG. 3 is a schematic plan view of a culture tank 40 that constitutes the artificial skin tissue manufacturing apparatus 30. FIG. It is a sectional view showing a manufacturing method of artificial skin tissue 1 concerning a 1st embodiment. It is a sectional view showing a manufacturing method of artificial skin tissue 1 concerning a 1st embodiment. It is a sectional view showing a manufacturing method of artificial skin tissue 1 concerning a 1st embodiment. It is a sectional view showing a manufacturing method of artificial skin tissue 1 concerning a 1st embodiment. It is a sectional view showing a manufacturing method of artificial skin tissue 1 concerning a 1st embodiment. It is a sectional view showing a manufacturing method of artificial skin tissue 1 concerning a 1st embodiment. FIG. 10 is a photographic view of observation of sensory nerve cells 14 in culture. FIG. 10 is a diagram showing a tissue-stained image showing sensory neurons and neurites after fixation to pre-gel body GP. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a cross-sectional stained image of a neurite produced by the production method of the second embodiment using a first sample of only spheroids 15. FIG. 4 is a stained image of a section of artificial skin tissue manufactured using the first sample by the manufacturing method of the second embodiment. This is a stained image of spheroids. It is an immunostaining image when culturing spheroids with different mixing ratios of sensory neurons and Schwann cells. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 3rd Embodiment. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 3rd Embodiment. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 4th Embodiment. It is sectional drawing which shows the manufacturing method of the artificial skin tissue 1 which concerns on 4th Embodiment. It is a cross-sectional view showing a method for measuring the artificial skin tissue 1 of the present invention. 6 is an enlarged view schematically showing an electrode map, electrode positions and addresses in an array chip 61. FIG. FIG. 4 is a diagram showing the relationship between time and voltage measured at electrodes of each address. FIG. 4 is a diagram showing the relationship between time and amplitude measured at electrodes of each address;

Embodiments of a method for producing an artificial three-dimensional tissue, a method for measuring an artificial three-dimensional tissue, and an artificial three-dimensional tissue according to the present invention will be described below with reference to FIGS. 1 to 26. FIG. Artificial three-dimensional tissue is artificial skin tissue.
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. Also, in the drawings below, in order to make each configuration easier to understand, the actual structure and each structure are different in scale, number, and the like.

(artificial skin tissue)
First, an 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.
Artificial skin tissue 1 includes dermal tissue layer 10 and epidermal tissue layer 20 . The artificial skin tissue 1 is arranged in a predetermined direction (horizontal direction in FIG. 1) along a plane perpendicular to the direction in which the dermal tissue layer 10 and the epidermal tissue layer 20 are laminated (vertical direction in FIG. 1, hereinafter referred to as the lamination direction). ).

The epidermal tissue layer 20 is formed by seeding and culturing epidermal cells 21 on the dermal tissue layer 10 . As the epidermal cells 21, for example, epidermal keratinocytes can be used.

Epidermal cells include cells derived from mammals such as humans, mice, and rats, with human-derived epidermal cells being preferred. Human-derived epidermal cells include epidermal keratinocytes and epidermal melanocytes, with epidermal keratinocytes being preferred. Epidermal keratinocytes include normal human epidermal keratinocytes (NHEK). Epidermal melanocytes include normal human epidermal melanocytes (NHEM).

Dermal tissue layer 10 is formed by culturing dermal cells 12 in extracellular matrix component 11, and includes first culture 12A in which dermal cells 12 are cultured. The extracellular matrix component 11 is not particularly limited, but examples include collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, heparan sulfate proteoglycan, etc. ), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, etc. reconstituted from basement membrane components (trade name: Matrigel). Fibroblasts, for example, can be used as the dermal cells 12 .

Fibroblasts include cells derived from mammals such as humans, mice, and rats, with human-derived fibroblasts being preferred. Human-derived fibroblasts include normal human dermal fibroblasts (NHDF), human lung fibroblasts: Human Pulmonary Fibroblasts (HPF), human cardiac fibroblasts: Human Cardiac Fibroblasts (HCF), human aorta Adventitial fibroblasts: Human Aortic Adventitial Fibroblasts (HAoAF), human uterine fibroblasts: Human Uterine Fibroblasts (HUF), human villous mesenchymal fibroblasts: Human Villous Mesenchymal Fibroblasts (HVMF), etc., human skin Fibroblasts (NHDF) are preferred.

The dermal tissue layer 10 contains spheroids 15 densely packed with sensory nerve cells 14 and neurites 16 .
The sensory nerve cells 14 are not particularly limited, but are derived from spinal ganglia of humans, mice and rats, brains (cortex/hippocampus/midbrain/hindbrain) of humans, mice and rats, and derived from human iPS cells. be able to. In this embodiment, it is derived from E15 mice.

The neurites 16 extend from within the dermal tissue layer 10 to the epidermal tissue layer 20 . The neurites 16 extend from the dermal tissue layer 10 containing the spheroids 15 toward the epidermal tissue layer 20 . That is, the dermal tissue layer 10 contains sensory nerve cells 14 and neurites 16 as a second culture medium 15A in which the spheroids 15 are cultured.

(Artificial skin tissue manufacturing device)
Next, an artificial skin tissue manufacturing apparatus for manufacturing the artificial skin tissue 1 will be described with reference to FIGS. 2 and 3. FIG.
FIG. 2 is a schematic plan view of a culture tank 40 that constitutes the artificial skin tissue manufacturing apparatus 30. As shown in FIG. FIG. 3 is a longitudinal sectional view of the artificial skin tissue manufacturing apparatus 30. As shown in FIG.
As shown in FIGS. 2 and 3 , the artificial skin tissue manufacturing apparatus 30 has a culture tank 40 and a culture dish 50 .

The culture tank 40 is formed in a square tubular shape that is rectangular in plan view. The culture tank 40 has a side wall 41 , a culture space 42 and an anchor portion 43 . The side wall 41 has a rectangular frame shape. The culture space 42 is surrounded by the side wall 41 and vertically penetrates the culture tank 40 . The anchor part 43 is provided so as to protrude from the inner wall surface 41 a of the side wall 41 into the culture space 42 . A plurality of (three in FIG. 2) anchor portions 43 are provided at intervals in the length direction of the side wall 41 for each side wall 41 forming each side of the rectangular frame. The anchor portions 43 provided on the opposing side walls 41 are arranged to face each other. As shown in FIG. 3 , the anchor part 43 protrudes into the culture space 42 from the middle position in the height direction of the side wall 41 along the normal direction of the inner wall surface 41 a. A direction parallel to the normal direction of the inner wall surface 41a and in which the anchor portions 43 face each other across the culture space 42 is called a facing direction.

The anchor portion 43 has a first portion 43A and a second portion 43B. The first portion 43A has a cylindrical shape with a proximal end positioned on the inner wall surface 41a and protruding into the culture space 42 along the facing direction. The second portion 43B is provided at the tip of the first portion 43A. The second portion 43B has a columnar (disk) shape protruding from the tip of the first portion 43A into the culture space 42 along the opposing direction. The outer diameter dimension of the second portion 43B is larger than the outer diameter dimension of the first portion 43A. The second portion 43B has an engaging surface 44 facing the inner wall surface 41a. In the culture space 42, a region surrounded by the inner wall surface 41a, the outer peripheral surface of the first portion 43A, and the engaging surface 44 serves as an anchor space. A culture body (details of which will be described later) located in the anchor space is restrained from contracting in the opposite direction by engaging with the engaging surface 44 during contraction.

The culture tank 40 is manufactured, for example, by printing acrylic resin with a 3D printer and curing it with ultraviolet irradiation. The culture vessel 40 thus manufactured is preferably coated with parylene or the like, which is resistant to chemical substances and ultraviolet rays and has high biocompatibility.

The culture tank 40 is mounted on the culture dish 50 . The culture space 42 is formed by being surrounded by the side walls 41 and the culture dish 50 . The culture dish 50 is provided with a holding portion 51 . The holding portion 51 holds the outer wall surface 41b of the side wall 41 from the outside. By holding the outer wall surface 41 b with the holding part 51 from the outside, it is possible to suppress leakage of the sol from the culture space 42 . As an example, the holding part 51 is manufactured by pouring polydimethylsiloxane (PDMS) around the culture tank 40 and baking it for a predetermined time.

(Method for producing artificial skin tissue 1)
Next, a method for manufacturing the artificial skin tissue 1 as a method for manufacturing an artificial three-dimensional tissue will be described with reference to FIGS. 3 to 8. FIG.
In the method for producing an artificial skin tissue (artificial three-dimensional tissue) 1 according to the present invention, a culture tank 40 having a culture space 42 surrounded by side walls 41 and anchor portions 43 provided on the opposing side walls 41 is prepared. and a gel body having a first culture body 12A in which dermal cells 12 are cultured by culturing in a culture tank 40 and a second culture body 15A in which spheroids 15 densely packed with sensory nerve cells 14 are cultured. and culturing the epidermal cells 21 arranged on the gel body G.

A method for manufacturing the artificial skin tissue 1 will be described in detail below.
[First embodiment of method for producing artificial skin tissue 1]
First, a first embodiment of the method for manufacturing the artificial skin tissue 1 will be described.
(Preparation of culture tank 40)
As preparation for the culture tank 40, the culture tank 40 having the side wall 41 provided with the anchor part 43 is placed on the culture dish 50, and the outer wall surface 41b is held by the holding part 51 so that the culture tank 40 is attached to the culture dish. Let it hold at 50.

(Acquisition of gel body G)
In obtaining the gel body G in the first embodiment, first, as shown in FIG. Pour into space 42 . The mixture is poured in such an amount that the anchor portion 43 is immersed. In this embodiment, collagen is used as the extracellular matrix component 11 .

When the mixture of the extracellular matrix component 11 and the dermal cells 12 is poured into the culture tank 40, the mixture is cultured (incubated) under predetermined conditions. The culture conditions are such that the density of the dermal tissue layer 10 is equivalent to that of human dermis. As an example of culture conditions, culture is performed 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 located in the anchor space described above engages the engagement surface 44 during contraction, as shown in FIG. 4, contraction in the stacking direction is not constrained, but contraction in the opposing direction is constrained. . As a result, a pre-gel body GP (dermal tissue layer 10) containing the first culture body 12A of cell bodies in which the dermal cells 12 are cultured can be obtained. Although not shown, the pre-gel body GP is immersed in a culture medium.

Next, as shown in FIG. 5, a well W is placed on the first surface Ga, which is the upper surface of the pregel body GP, and spheroids 15 of sensory nerve cells 14 are seeded inside the well W together with a medium M. placed and cultured. 5 to 8, the illustration of the culture dish 50 and the holding part 51 is omitted, but the second surface Gb of the pre-gel body GP opposite to the first surface Ga is immersed in the culture medium. there is

By culturing the spheroids 15 on the first surface Ga of the pre-gel body GP, as shown in FIG. A body 15A is provided.
As a result, a gel body G including a dermal tissue layer 10 having a first cultured body 12A of cell bodies in which dermal cells 12 are cultured and a second cultured body 15A in which spheroids 15 of sensory nerve cells 14 are cultured is obtained. be able to.

Next, the epidermal tissue layer 20 is formed. First, the culture tank 40 is turned upside down so that the second surface Gb is the upper surface and the first surface Ga is the lower surface, as shown in FIG. Next, a well W is placed on the second surface Gb which is the upper surface of the pre-gel body GP, and epidermal cells 21 (for example, normal human epidermal keratinocytes) are seeded inside the well W together with a medium M. Arrange and culture. As a result, the epidermal cells 21 are induced to differentiate, and an epidermal tissue layer 20 can be formed as shown in FIG. As an example of culture conditions for forming the epidermal tissue layer 20, the temperature is 37° C. for 9 days.

The artificial skin tissue having the second culture 15A on the first surface Ga of the gel body G of the dermis tissue layer 10 containing the first culture 12A and the epidermal tissue layer 20 on the second surface Gb by the above manufacturing method. 1 is obtained.

FIG. 9 is a photographic diagram showing observation of sensory neurons in culture. (a) in FIG. 9 is a bright-field observation image, and (b) is a fluorescence observation image. As shown in FIGS. 9(a) and 9(b), it was possible to observe that neurites were extending even on the surface of the artificial skin tissue from the spread of cells around the spheroids. When the spheroid into which the red fluorescent protein was introduced was observed, as shown in (b), red fluorescence was confirmed, confirming that the observed tissue was a spheroid of sensory nerve cells.

In the above manufacturing method, the culture tank 40 was turned upside down when forming the epidermal tissue layer 20, so the spheroids 15 were in a state of hanging from the pre-gel body GP. There was no detachment from body GP.
It was confirmed that the state of the sensory nerve cells 14 on the artificial skin tissue 1 could be observed while being cultured by using the above manufacturing method.

FIG. 10 shows sensory nerve cells and neurites fixed to gel body G, (c) is a stained image of a section, and (d) is a diagram showing a tissue stained image.
As shown in FIG. 10 , when spheroids 15 were seeded in the pre-gel body GP, neurites 16 were observed to extend mainly on the surface of dermal tissue layer 10 . It is considered that this is because the pre-gel body GP contracts in the opposite direction, so that the dermal cells 12 are oriented in the opposite direction during contraction.

[Second embodiment of method for producing artificial skin tissue 1]
Next, a second embodiment of the method for manufacturing the artificial skin tissue 1 will be described with reference to FIGS. 11 to 18. FIG. In these figures, the same reference numerals are given to the same elements as those of the first embodiment shown in FIGS. 1 to 8, and the description thereof will be omitted or simplified.

(Acquisition of gel body G)
In obtaining the gel body G in the second embodiment, the dermal cells 12 in the extracellular matrix component 11 and the spheroids 15 of the sensory nerve cells 14 are cultured to obtain the first culture body 12A and the second culture body 15A. A gel body containing therein is obtained.

Specifically, as shown in FIG. 11, a mixture of extracellular matrix components 11, dermal cells 12 (for example, normal human dermal fibroblasts), and spheroids 15 of sensory nerve cells 14 is cultured in a culture tank 40. Pour into space 42 . The mixture is poured in such an amount that the anchor portion 43 is immersed.

For the spheroids 15 in this embodiment, a first sample in which sensory nerve cells 14 are aggregated and cultured alone and a second sample in which an established Schwann cell line is mixed and cultured are used. The extracellular matrix component 11 and the spheroids 15 mixed with the dermal cells 12 are individually selected and cultured when using the first sample and when using the second sample.

After the mixture of the extracellular matrix component 11, the dermal cells 12 and the spheroids 15 is poured into the culture tank 40, the mixture is cultured (incubated) under predetermined conditions.

The extracellular matrix component 11 contracts by culturing. Since the extracellular matrix component 11 located in the anchor space described above engages the anchor portion 43 during contraction, as shown in FIG. 12, contraction in the stacking direction is not constrained, but contraction in the opposing direction is constrained. As a result, a gel body G (dermal tissue layer 10) containing a first cultured body 12A of cell bodies in which dermal cells 12 are cultured and a second cultured body 15A in which spheroids 15 of sensory nerve cells 14 are cultured is obtained. can be done. Although not shown, the lower surface of the gel body G is immersed in a culture medium.

Next, as shown in FIG. 13, wells W are placed on the first surface Ga, which is the upper surface of the gel body G, and epidermal cells 21 (for example, normal human epidermal keratinocytes) are placed inside the wells W. Inoculate with medium M, arrange and culture. As a result, the epidermal cells 21 are induced to differentiate, and as shown in FIG. 14, the epidermal tissue layer 20 can be formed. As an example of culture conditions for forming the epidermal tissue layer 20, the temperature is 37° C. for 9 days.

By the above manufacturing method, the artificial skin tissue 1 having the epidermal tissue layer 20 on the first surface Ga of the gel body G of the dermal tissue layer 10 including the first cultured body 12A and the second cultured body 15A is obtained.

FIG. 15 is a cross-sectional stained image of artificial skin tissue produced by the production method of the second embodiment using the first sample of spheroids 15 only. In FIG. 15, image (e') is an enlarged view of image (e). FIG. 16 is a stained image of a section of the artificial skin tissue produced by the production method of the second embodiment using the first sample.
As shown in FIGS. 15 and 16, when the spheroids 15 were placed inside the gel body G of the dermal tissue layer 10, neurite extension was observed up to about half the thickness of the dermal tissue layer 10.

FIG. 17 shows staining images of spheroids, (f) is a staining image of the first sample cultured only with spheroids, and (g) is a staining image of the second sample cultured by mixing spheroids and the established Schwann cell line. is a statue.
As shown in FIG. 17, it was confirmed that neurite extension was greater when the established Schwann cell line was incorporated into the spheroid.
Therefore, by using the second sample of spheroids into which the established Schwann cells are incorporated, the artificial skin tissue is produced by the production method of the second embodiment, until the nerve cells reach the epidermal tissue layer from the dermal tissue layer. It is thought that it will expand.

FIG. 18 is an immunostaining image when spheroids were cultured with different mixing ratios of sensory neurons and Schwann cells. As for the mixing ratio of sensory neurons and Schwann cells, [number of sensory neurons:number of Schwann cells] is preferably in the range of 3:1 to 1:3, preferably 1:1 to 1:3. is more preferable.
FIG. 18 shows a stained image using only sensory neurons, a stained image when the number of sensory neurons and the number of Schwann cells is 1:1 (10000:10000), and the number of sensory neurons and the number of Schwann cells. Stained image when the ratio is 1:2 (6700:13300), stained image when the number of sensory neurons and Schwann cells is 1:3 (5000:15000), and the number of sensory neurons and Schwann cells A stained image is shown when the number is 1:4 (2500:17500).
As shown in FIG. 18, culturing by mixing the number of sensory nerve cells and the number of Schwann cells in the range of 1:1 to 1:3 resulted in greater neurite extension and the effect of incorporating established Schwann cells. I can confirm that it is high.

[Third embodiment of method for producing artificial skin tissue 1]
Next, a third embodiment of the method for manufacturing the artificial skin tissue 1 will be described with reference to FIGS. 19 to 20. FIG. In these figures, the same reference numerals are assigned to the same elements as those of the second embodiment shown in FIGS. 11 to 18, and the description thereof will be omitted or simplified.

As shown in FIG. 19 , in the manufacturing method of the present embodiment, the preparation of the culture tank 40 includes installing linear holding members 45 along the opposing direction between the opposing anchor portions 43 .
The material of the holding member 45 is not particularly limited as long as it does not adversely affect the culture of the artificial skin tissue 1, but is preferably a material to which the spheroids 15 easily adhere when cultured in the extracellular matrix component 11.

In the production method of the second embodiment, when culturing the mixture of the extracellular matrix component 11, the dermal cells 12, and the spheroids 15, the spheroids 15 settle due to their own weight. Therefore, it is considered that the distance between the spheroids 15 and the epidermal tissue layer 20 in the artificial skin tissue 1 becomes longer, and the neurites extending from the spheroids 15 are less likely to reach the epidermal tissue layer 20 .

In this embodiment, since the linear holding members 45 are installed along the opposing direction between the anchor portions 43, at least a part of the spheroids 15 that settle when culturing the mixture adheres to the holding members 45. and retained. As shown in FIG. 20 , the retained spheroids 15 are located near the center of the dermal tissue layer 10 in the height direction, so the neurites extending from the spheroids 15 are located on the lower surfaces of the epidermal tissue layer 20 and the dermal tissue layer 10 . Easier to reach both.

[Fourth Embodiment of Method for Manufacturing Artificial Skin Tissue 1]
Next, a fourth embodiment of the method for manufacturing the artificial skin tissue 1 will be described with reference to FIGS. 21 to 22. FIG. In these figures, the same reference numerals are assigned to the same elements as those of the first embodiment shown in FIGS. 3 to 8, and the description thereof will be omitted or simplified.

In the manufacturing method of this embodiment, the linear member 46 having a diameter larger than the maximum diameter of the spheroid 15 and smaller than the outer diameter of the first portion 43A of the anchor portion 43 is used. The linear member 46 is coaxial with the anchor portion 43, passes through the anchor portion 43 and the side wall 41, and is provided to be insertable and removable.

Pre-gel body GP (dermal tissue layer 10) is obtained by pouring a mixture of extracellular matrix component 11 and dermal cells 12 into culture tank 40 and culturing. As a result, the pre-gel body GP through which the linear members 46 penetrate in the opposing direction is obtained.
After the pre-gel body GP is obtained, the linear member 46 is removed to form a cavity 46A penetrating the pre-gel body GP in the opposite direction, as shown in FIG. The cavity 46A is located near the center of the dermal tissue layer 10 in the height direction.

Next, the spheroids 15 are seeded together with a medium in the cavity 46A of the pre-gel body GP, arranged and cultured. After placing the spheroids 15 together with the culture medium in the cavity 46A, the plug 43C is embedded to prevent leakage of the spheroids 15 and the culture medium. Then, by culturing the spheroids 15, a second culture body 15A containing the spheroids 15 is provided.

In the present embodiment, in order to culture the spheroids 15 in the cavity 46A located near the center of the dermal tissue layer 10 in the height direction, the spheroids 15 are cultured near the center of the dermis tissue layer 10 in the height direction, as in the third embodiment. Located in
Therefore, by using the manufacturing method of the present embodiment, the neurites extending from the spheroid 15 can easily reach both the lower surfaces of the epidermal tissue layer 20 and the dermal tissue layer 10 .

[Method for measuring artificial skin tissue 1]
In the method for measuring the artificial skin tissue 1, the extracellular potential of the artificial skin tissue 1 obtained by the manufacturing method described above is measured. FIG. 23 is a cross-sectional view showing a method for measuring the artificial skin tissue 1. FIG. The extracellular potential is measured using a measuring instrument 60, as shown in FIG. A measuring instrument 60 has an array chip 61 on the top surface of which a plurality of microelectrodes are arranged in an array. The potential measured by the microelectrodes in the array chip 61 is output to a control section (not shown).

A method for measuring the artificial skin tissue 1 includes preparing the measuring instrument 60 and placing the artificial skin tissue 1 manufactured by the manufacturing method described above on an array chip 61 (microelectrode). The method for measuring the artificial skin tissue 1 further includes placing the artificial skin tissue 1 on the array chip 61 and pressing the artificial skin tissue 1 toward the array chip 61 . The artificial skin tissue 1 is pressed toward the array chip 61 by driving the holder 62 placed on the upper side of the artificial skin tissue 1 downward. By driving the holder 62 downward, the adhesion between the array chip 61 and the spheroids 15 in the artificial skin tissue 1 is enhanced, and the extracellular potential measurement accuracy can be enhanced.

FIG. 24 is an enlarged view schematically showing an electrode map, electrode positions and addresses in the array chip 61. As shown in FIG. FIG. 24 shows the state in which potentials are measured at the electrodes of addresses 8434-8436 and 8654-8656.

FIG. 25 is a diagram showing the relationship between time and voltage measured at the electrodes of each address.
FIG. 26 is a diagram showing the relationship between time and amplitude measured at each address electrode.
As shown in FIG. 25, it was confirmed that a spike-like signal was supplied to the electrodes. From the above results, synchronous spike waveforms were measured around a specific electrode, suggesting that sensory nerve activity could be measured, and that the function of sensory nerve cells in the artificial skin tissue 1 could be measured. It is suggested.
Therefore, by using the method for measuring the artificial skin tissue 1 of the present embodiment, it is possible to easily perform functional analysis of sensory nerve cells.

(Drug evaluation)
As still another aspect of the present invention, the irritation of a drug to the artificial skin tissue 1 can be evaluated. The evaluation of the drug can be performed, for example, by bringing the drug into contact with the artificial skin tissue 1 and measuring the response to the stimulation caused by the contact of the drug using the measurement method described above.
The drug in the present invention includes drugs such as pharmaceuticals, cosmetics, quasi-drugs, and the like. According to the evaluation method of the present invention, for example, a drug can be evaluated in an environment closer to the actual artificial skin tissue 1 than in conventional methods. In addition, the evaluation method of the present invention is extremely useful, for example, in evaluating the kinetics of drugs of various molecular weights in the creation (screening) of new drugs, and in the evaluation in the development of cosmetics, quasi-drugs, and the like. A drug is a substance to be evaluated, and examples thereof include inorganic compounds and organic compounds.

Although the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, it goes without saying that the present invention is not limited to such examples. The various shapes, combinations, etc., of the constituent members shown in the above examples are merely examples, and various modifications can be made based on design requirements and the like without departing from the gist of the present invention.

For example, in the method for measuring the artificial skin tissue 1, the procedure of placing the artificial skin tissue 1 manufactured by the manufacturing method described above on the array chip 61 was exemplified, but the configuration is not limited to this. For example, in the manufacturing method described above, preparing the culture vessel 40 may include preparing the measuring instrument 60 and placing the culture vessel 40 on the array chip 61 (microelectrode). In this case, the gel body G is cultured in a culture tank 40 and has a first culture body 12A in which dermal cells 12 are cultured and a second culture body 15A in which spheroids 15 densely packed with sensory nerve cells 14 are cultured. Obtaining and culturing the epidermal cells 21 arranged on the gel body G may be performed on the array chip 61 (microelectrode).
By using this manufacturing method, the functional analysis of the sensory nerve cells can be easily performed in the manufacturing process of the artificial skin tissue 1 without performing the procedure of mounting the artificial skin tissue 1 on the array chip 61 .

Further, although the holding member 45 shown in the above embodiment is a linear member that does not adversely affect the culture of the artificial skin tissue 1, it is not limited to this configuration. For example, a configuration using fibers with or containing spheroids 15 made of a gel material or polymer may be used.
By adopting this configuration, in addition to the spheroids 15 that have sedimented during culture and adhered to the holding member 45, neurites extending from the spheroids 15 provided on the holding member 45 are formed in the epidermal tissue layer 20 and the dermal tissue layer 10. It becomes easier to reach both sides of the lower surface.

It should be noted that the inventions described in the above embodiments are arranged and disclosed as appendices.
(Appendix 1)
preparing a culture tank having a culture space surrounded by side walls and anchor portions provided on the opposing side walls; and performing culture in the culture tank to culture the dermal cells. and a second culture body in which spheroids densely packed with sensory nerve cells are cultured; and culturing epidermal cells arranged on the gel body. Production method.

(Appendix 2)
Obtaining the gel body having the first culture body and the second culture body includes:
culturing the dermal cells in the extracellular matrix component to obtain a pre-gel containing the first culture;
culturing the spheroids arranged on the first surface of the pre-gel body to obtain the pre-gel body having the second culture body on the first surface;
culturing the epidermal cells arranged on the second surface opposite to the first surface of the pre-gel body having the second culture body;
including,
A method for producing an artificial three-dimensional tissue according to appendix 1.

(Appendix 3)
Obtaining the gel body having the first culture body and the second culture body includes:
culturing the dermal cells and the spheroids in the extracellular matrix component to obtain the gel body containing the first culture body and the second culture body therein;
A method for producing an artificial three-dimensional tissue according to appendix 1.

(Appendix 4)
Preparing the culture tank includes installing a linear holding member along the opposing direction between the opposing anchor parts,
A method for producing an artificial three-dimensional tissue according to appendix 3.

(Appendix 5)
Preparing the culture tank includes installing a linear member along the opposing direction between the opposing anchor portions,
Obtaining the gel body having the first culture body and the second culture body includes:
obtaining a pre-gel body containing the first culture body by culturing the dermal cells in the extracellular matrix component and having the linear members penetrate in the opposite direction;
removing the linear member from the pre-gel body to form a cavity penetrating the pre-gel body in the opposite direction;
obtaining the second culture by culturing the spheroid placed in the cavity;
including,
A method for producing an artificial three-dimensional tissue according to appendix 1.

(Appendix 6)
obtaining the second culture by culturing the spheroids and Schwann cells;
The method for producing an artificial three-dimensional tissue according to any one of Appendixes 1 to 5.

(Appendix 7)
The number of sensory nerve cells and the number of Schwann cells are mixed and cultured in a ratio range of 3: 1 to 1: 3.
A method for producing an artificial three-dimensional tissue according to appendix 6.

(Appendix 8)
Preparing the culture tank includes installing a measuring instrument in which a plurality of electrodes are arranged in an array and measures an extracellular potential with the electrodes,
obtaining the gel body and culturing the epidermal cells on the electrode;
The method for producing an artificial three-dimensional tissue according to any one of appendices 1 to 7.

(Appendix 9)
Preparing a measuring instrument in which a plurality of electrodes are arranged in an array for measuring an extracellular potential with the electrodes;
placing the artificial three-dimensional tissue produced by the method for producing an artificial three-dimensional tissue according to any one of appendices 1 to 8 on the electrode;
A method for measuring an artificial three-dimensional tissue, comprising:

(Appendix 10)
pressing the artificial three-dimensional tissue placed on the electrode toward the electrode;
The method for measuring an artificial three-dimensional tissue according to appendix 9.

(Appendix 11)
bringing a drug into contact with the artificial three-dimensional tissue;
Evaluating the agent by measuring the response of the artificial three-dimensional tissue to stimulation by contact with the agent;
including,
The method for measuring an artificial three-dimensional tissue according to appendix 9 or appendix 10.

(Appendix 12)
a dermal tissue layer comprising dermal cells;
an epidermal tissue layer containing epidermal cells and formed on the dermal tissue layer;
Spheroids contained in the dermal tissue layer and densely packed with sensory nerve cells having neurites;
An artificial three-dimensional tissue having

1 Artificial skin tissue (artificial three-dimensional tissue) 10 Dermal tissue layer 11 Extracellular matrix component 12 Dermal cell 12A First culture 14 Sensory nerve cell 15 Spheroid 15A Second 2 cultures 16 neurites 20 epidermal tissue layer 21 epidermal cells 40 culture tank 41 side wall 42 culture space 43 anchor part 45 holding member 46 linear member 60... Measuring instrument G... Gel body Ga... First surface Gb... Second surface GP... Pre-gel body

Claims (12)

Preparing a culture vessel having a culture space surrounded by side walls and anchor portions provided on the opposing side walls;
Culturing in the culture tank to obtain a gel body having a first culture body in which dermal cells are cultured and a second culture body in which spheroids densely packed with sensory nerve cells are cultured;
culturing the epidermal cells arranged on the gel body;
A method for producing an artificial three-dimensional tissue, comprising: Obtaining the gel body having the first culture body and the second culture body includes:
culturing the dermal cells in the extracellular matrix component to obtain a pre-gel containing the first culture;
culturing the spheroids arranged on the first surface of the pre-gel body to obtain the pre-gel body having the second culture body on the first surface;
culturing the epidermal cells arranged on the second surface opposite to the first surface of the pre-gel body having the second culture body;
including,
The method for producing an artificial three-dimensional tissue according to claim 1. Obtaining the gel body having the first culture body and the second culture body includes:
culturing the dermal cells and the spheroids in the extracellular matrix component to obtain the gel body containing the first culture body and the second culture body therein;
The method for producing an artificial three-dimensional tissue according to claim 1. Preparing the culture tank includes installing a linear holding member along the opposing direction between the opposing anchor parts,
The method for producing an artificial three-dimensional tissue according to claim 3. Preparing the culture tank includes installing a linear member along the opposing direction between the opposing anchor portions,
Obtaining the gel body having the first culture body and the second culture body includes:
obtaining a pre-gel body containing the first culture body by culturing the dermal cells in the extracellular matrix component and having the linear members penetrate in the opposite direction;
removing the linear member from the pre-gel body to form a cavity penetrating the pre-gel body in the opposite direction;
obtaining the second culture by culturing the spheroid placed in the cavity;
including,
The method for producing an artificial three-dimensional tissue according to claim 1. obtaining the second culture by culturing the spheroids and Schwann cells;
A method for producing an artificial three-dimensional tissue according to any one of claims 1 to 5. culturing by mixing the number of sensory nerve cells and the number of Schwann cells in a ratio range of 3:1 to 1:3;
The method for producing an artificial three-dimensional tissue according to claim 6. Preparing the culture tank includes installing a measuring instrument in which a plurality of electrodes are arranged in an array and measures an extracellular potential with the electrodes,
obtaining the gel body and culturing the epidermal cells on the electrode;
A method for producing an artificial three-dimensional tissue according to any one of claims 1 to 5. Preparing a measuring instrument in which a plurality of electrodes are arranged in an array for measuring an extracellular potential with the electrodes;
placing the artificial three-dimensional tissue manufactured by the method for manufacturing an artificial three-dimensional tissue according to any one of claims 1 to 5 on the electrode;
A method for measuring an artificial three-dimensional tissue, comprising: pressing the artificial three-dimensional tissue placed on the electrode toward the electrode;
The method for measuring an artificial three-dimensional tissue according to claim 9. bringing a drug into contact with the artificial three-dimensional tissue;
Evaluating the agent by measuring the response of the artificial three-dimensional tissue to stimulation by contact with the agent;
including,
The method for measuring an artificial three-dimensional tissue according to claim 9. a dermal tissue layer comprising dermal cells;
an epidermal tissue layer containing epidermal cells and formed on the dermal tissue layer;
Spheroids contained in the dermal tissue layer and densely packed with sensory nerve cells having neurites;
An artificial three-dimensional tissue having

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