JP7388724B2 - Artificial multilayer tissue culture device and method for producing artificial multilayer tissue - Google Patents

Description

The present invention relates to an artificial multilayer tissue culture device and a method for manufacturing an artificial multilayer tissue.
This application claims priority from U.S. Provisional Patent Application No. 62/751,743, filed October 29, 2018, the contents of which are hereby incorporated by reference.

In an artificial multilayer tissue such as a skin model, each layer has a large thickness in the plane direction. Therefore, in order to observe the cross section of the artificial multilayer tissue, it is necessary to cut the artificial multilayer tissue and prepare tissue sections. However, this work requires a lot of time and cost. Furthermore, there is a problem in that the sample is easily broken when tissue sections are prepared. Furthermore, since the artificial multilayer tissue is cut, there is a problem in that it is not possible to observe the passage of time using the same sample.

Non-Patent Document 1 and Non-Patent Document 2 disclose a device in which a medium channel is provided adjacent to both sides of a gel channel, and cells are added from the medium channel to the gel channel and cultured.

In the devices disclosed in Non-Patent Document 1 and Non-Patent Document 2, since the gel flow path and the medium flow path are formed on the same plane, cells can be observed without cutting by observing in the normal direction of the plane. It becomes possible to observe.

MEDICAL SCIENCES, ENGINEERING “Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation,” by Jessie S. Jeon, Simone Bersini, Mara Gilardi, Gabriele Dubini, Joseph L. Charest, Matteo Moretti, and Roger D. Kamm, which appeared in issue 1, January 6, 2015, of Proc Natl Acad Sci USA(112:214－219; first published December 18, 2014; 10.1073/pnas.1417115112) AIM BIOTECH, “TECHNOLOGY”, [Retrieved October 11, 2020], Internet <URL: https://www.aimbiotech.com/technology.html>

However, with the above-mentioned device, although it is possible to form and observe cell tissue (hereinafter referred to as gel channel tissue) in the gel channel, it is possible to form and observe a tissue composed of multilayered cells in the medium channel (hereinafter referred to as "gel channel tissue"). It is difficult to form a channel structure (channel structure), and it cannot be said to be suitable for observing an artificial multilayer structure composed of a gel channel structure and a medium channel structure. Furthermore, when the medium channel tissue is composed of epithelial cells, the above-described device has difficulty in culturing the tissue at the air-liquid interface, and is not suitable for controlling the differentiation of epithelial tissue.

The present invention was made in view of the above problems, and aims to provide an artificial multilayer tissue culture device and a method for manufacturing an artificial multilayer tissue that allow observation of the passage of time using the same sample without creating tissue sections. do.

According to the first aspect of the present invention, the first flow path is opened on one surface of the first substrate and extends in the first direction, and in which the first cells are cultured; a second channel extending in the direction in which the second cells are cultured; a third channel opening in the one surface and extending in the first direction to which at least a medium is supplied; and a third channel in the first direction connected to the one surface. , a second substrate having optical transparency that seals the second flow path and the third flow path, and the second flow path is arranged in the first direction with respect to the first flow path. provided on one side of the intersecting second direction via a first partition, and the third flow path is provided on the other side of the second direction with respect to the first flow path via a second partition, The first partition wall has a first communication path that connects the first flow path and the second flow path, and the second partition wall connects the first flow path and the third flow path. An artificial multilayer tissue having a second communicating path, wherein the first communicating path has an opposing surface that faces the first direction and tapers from the second flow path to the first flow path. A culture device is provided.

According to a second aspect of the present invention, providing an artificial multilayer tissue culture device according to the first aspect of the present invention, and culturing a first cell in the first channel to form a first tissue layer. supplying the second cells and culture medium to the second flow path and supplying the culture medium to the third flow path; adjusting the posture of the artificial multilayer tissue culture device; locating the second channel above the channel, arranging the second cell on the first tissue layer exposed to the first communication channel, and disposing the second cell on the first tissue layer, There is provided a method for producing an artificial multilayer tissue, comprising: culturing cells in the medium in the second channel.

According to the present invention, it is possible to provide an artificial multilayer tissue culture device and a method for manufacturing an artificial multilayer tissue that allow observation of the passage of time using the same sample without creating tissue sections. According to the present invention, for example, it is possible to construct and observe a multilayered tissue having both epithelial tissue and endothelial tissue.

FIG. 1 is a schematic perspective view of an artificial multilayer tissue observation system SYS having an artificial multilayer tissue culture device of the present invention. FIG. 2 is a plan view of the first substrate 10 viewed from above. FIG. 3 is a cross-sectional view taken along the line AA in FIG. 2. FIG. 3 is a partial plan view of the vicinity of first portions 13B and 13C viewed from the -Z side. FIG. 2 is a partial plan view of an artificial multilayer tissue cultured and formed using the artificial multilayer tissue culture device 1, viewed from the -Z side. FIG. 5 is a partial plan view showing the manufacturing process of the artificial multilayer tissue 50. FIG. FIG. 5 is a partial plan view showing the manufacturing process of the artificial multilayer tissue 50. FIG. FIG. 5 is a partial plan view showing the manufacturing process of the artificial multilayer tissue 50. FIG. This is an image taken of a state in which 10-50 cells of epidermal cells 71 are arranged (seeded) on a dermal tissue layer 60. This is an image taken of the dermal tissue layer 60 and the epidermal layer 70 in the first communicating path 16 after culturing 10-50 cells of epidermal cells 71 for one day. This is a fluorescence image taken of the same dermal tissue layer 60 and epidermal layer 70. This is a fluorescence image taken in the Y direction of the epidermal layer 70. FIG. 2 is a schematic perspective view of an artificial multilayer tissue observation system SYS having an artificial multilayer tissue culture device 1 according to a second embodiment of the present invention. FIG. 7 is a plan view of the first substrate 10 of the second embodiment viewed from above. FIG. 5 is a partial plan view showing the manufacturing process of the artificial multilayer tissue 50. FIG. FIG. 2 is a partial plan view of an artificial multilayer tissue 50 cultured and formed using the artificial multilayer tissue culture device 1, viewed from the -Z side.

EMBODIMENT OF THE INVENTION Hereinafter, embodiments to which the artificial multilayer tissue culture device and the method for manufacturing an artificial multilayer tissue of the present invention are applied will be described in detail with reference to the drawings. Note that the drawings used in the following explanation are for explaining the configuration of the embodiment of the present invention, and the size, thickness, dimensions, etc. of each part shown in the drawings may differ from the dimensional relationship of the actual toothbrush. be.

[Artificial multilayer tissue observation system]
FIG. 1 is a schematic perspective view of an artificial multilayer tissue observation system SYS having an artificial multilayer tissue culture device 1 according to a first embodiment of the present invention.
As shown in FIG. 1, the artificial multilayer tissue observation system SYS includes an artificial multilayer tissue culture device 1 and an observation device 2.

[First embodiment of artificial multilayer tissue culture device]
The artificial multilayer tissue culture device 1 includes a first substrate 10 and a second substrate 30, both of which have a rectangular plate shape in plan view. The first substrate 10 and the second substrate 30 are, for example, in the shape of a rectangular parallelepiped that are vertically joined using an adhesive or the like. The first substrate 10 is joined to the second substrate 30 at the lower surface (one surface) 10a.

The first substrate 10 and the second substrate 30 each have a side length of several mm to several tens of mm, for example. Further, the thickness of the first substrate 10 is, for example, several mm. The thickness of the second substrate 30 is, for example, several hundred μm.

The material of the first substrate 10 is not particularly limited, but examples thereof include elastomers such as silicone rubber and PDMS (polydimethylsiloxane). The first substrate 10 of this embodiment is made of PDMS from the viewpoint of air permeability.

The material of the second substrate 30 is not particularly limited, but examples include elastomer, silicone rubber, PDMS (polydimethylsiloxane), and glass. The second substrate 30 of this embodiment is made of PDMS from the viewpoint of light transmittance and air permeability.

FIG. 2 is a plan view of the first substrate 10 viewed from above.
As shown in FIG. 2, the first substrate 10 has a first channel FA that is provided approximately in the center in the width direction (in the left-right direction in FIG. 2) and in which the first cells are cultured, and A second channel FB is arranged on one side in the width direction (on the left side in FIG. 2) and where the second cells are cultured, and a third channel FB is arranged on the other side in the width direction (on the right side in FIG. 2) and where the third cells are cultured. It has a third flow path FC.

Note that in the following, the bonding direction between the first substrate 10 and the second substrate 30 (the normal direction of the lower surface 10a) is assumed to be the Z direction, and the first flow path FA, the second flow path FB, and the third flow path FC are lined up. In the following description, the width direction is referred to as the Y direction (second direction), and the Z direction and the direction orthogonal to the Y direction are referred to as the X direction (first direction).

The first flow path FA is formed to extend in the X direction. An inlet 11A is provided at the −X side end of the first flow path FA. An outlet 12A is provided at the +X side end of the first flow path FA. The inlet 11A and the outlet 12A each have a circular cross section, extend in the Z direction, and penetrate the first substrate 10.

An inlet 11B is provided at the -X side end of the second flow path FB. An outlet 12B is provided at the +X side end of the second flow path FB. The inlet 11B and the outlet 12B each have a circular cross section, extend in the Z direction, and penetrate the first substrate 10. The second flow path FB includes a first portion 13B disposed parallel to the first flow path FA along the X direction at the center of the X direction, and a second portion 14B disposed on both sides of the first portion 13B in the X direction. It has Each second portion 14B extends in a direction in which the distance in the Y direction from the first flow path FA increases as the distance from the first portion 13B in the X direction increases.

An inlet 11C is provided at the −X side end of the third flow path FC. An outlet 12C is provided at the +X side end of the third flow path FC. The inlet 11C and the outlet 12C each have a circular cross section, extend in the Z direction, and penetrate the first substrate 10. The third flow path FC includes a first portion 13C disposed along the X direction parallel to the first flow path FA at the center of the X direction, and a second portion 14C disposed on both sides of the first portion 13C in the X direction. It has Each second portion 14C extends in a direction in which the distance in the Y direction from the first flow path FA increases as the second portion 14C moves away from the first portion 13C in the X direction.

FIG. 3 is a cross-sectional view taken along line AA in FIG. FIG. 4 is a partial plan view of the vicinity of the first portions 13B and 13C viewed from the −Z side.
As shown in FIG. 3, the first flow path FA, the second flow path FB, and the third flow path FC are formed by grooves opening in the lower surface 10a of the first substrate 10. That is, the first flow path FA, the second flow path FB, and the third flow path FC are arranged in the same plane parallel to the XY plane.

The first portion 13B of the second flow path FB is provided to the first flow path FA via the first partition wall 15. As shown in FIG. 4, the first partition wall 15 has a first communication path 16 that communicates the first flow path FA with the first portion 13B of the second flow path FB. The first communicating path 16 is formed to penetrate the first partition wall 15 in the Y direction. The depth of the first communicating passage 16 is the same as the depth of the first passage FA and the second passage FB.

The first communicating path 16 has an opposing surface 17B located on the second flow path FB side and facing in the X direction, and a second opposing surface 18B located on the first flow path FA side and facing in the X direction. The opposing surface 17B tapers from the second flow path FB toward the first flow path FA. The second opposing surface 18B expands in diameter from the tapered end of the opposing surface 17B toward the first flow path side FA.

The intersecting angle between the opposing surfaces 17B is not particularly limited, but may be about 90 degrees, for example. The intersecting angle between the second opposing surfaces 18B is not particularly limited, but is preferably an angle at which the extracellular matrix component solution supplied to the first channel FA does not leak into the second channel FB due to surface tension, for example, 60 degree.

The wall surface of the first partition wall 15 facing the second flow path FB and the opposing surface 17B are subjected to surface treatment to suppress adhesion of second cells. As an example of the surface treatment, first, a solution of 0.2 wt% Pluronic F-127 dissolved in a serum-free medium is poured into the second channel and allowed to react for over 1 hour. After that, the cell suspension may be poured into the second channel without drying.

The first partition wall 15 has a first air storage portion 19B in a region separated from the first flow path FA, the second flow path FB, and the first communication path 16. As shown in FIG. 2, the first air storage section 19B is connected to an air inlet 25B that penetrates the first substrate 10 via an air introduction path 24B, and air is introduced therein. Since the PDMS forming the first substrate 10 has air permeability, the air contained in the first air storage section 19B passes through the first partition wall 15 to the first flow path FA and the second flow path. It can be supplied to FB and the first communication path 16. The first air storage portion 19B is provided with a plurality of pillar portions 20B having a rectangular cross section and extending in the Z direction. The plurality of pillar parts 20B are arranged at intervals in the X direction. The tip surface of the columnar portion 20B is flush with the lower surface 10a of the first substrate 10.

The first portion 13C of the third flow path FC is provided to the first flow path FA via the second partition wall 21. The second partition wall 21 has a second communication path 22 that communicates the first flow path FA with the first portion 13C of the third flow path FC. The second communicating path 22 is formed to penetrate the second partition wall 21 in the Y direction. The depth of the second communicating passage 22 is the same as the depth of the first passage FA and the third passage FC.

The second communicating path 22 has a facing surface 18C facing in the X direction. The opposing surface 18C tapers from the first flow path FA toward the third flow path FC. The intersecting angle between the opposing surfaces 18C is not particularly limited, but is preferably an angle at which the extracellular matrix component solution supplied to the first channel FA does not leak into the third channel FC due to surface tension, for example, 60 degrees. be.

The wall surface facing the second channel FB in the first partition 15 and the opposing surface 17B were subjected to surface treatment to suppress adhesion of second cells, but the wall surface facing the third channel FC in the second partition 21 was subjected to surface treatment to suppress adhesion of second cells. No surface treatment was applied to suppress the adhesion of third cells.

The second partition wall 21 has a second air storage section 19C in a region separated from the first flow path FA, the third flow path FC, and the second communication path 22. As shown in FIG. 2, the second air storage section 19C is connected to an air inlet 25C that penetrates the first substrate 10 via an air introduction path 24C, and air is introduced therein. The air accommodated in the second air storage section 19C can be supplied to the first flow path FA, the third flow path FC, and the second communication path 22 via the second partition wall 21. The second air storage portion 19C is provided with a plurality of pillar portions 20C having a rectangular cross section and extending in the Z direction. The plurality of column parts 20C are arranged at intervals in the X direction. The tip surface of the columnar portion 20C is flush with the lower surface 10a of the first substrate 10.

By being bonded to the lower surface 10a of the first substrate 10, the second substrate 30 is also bonded to the tip surfaces of the first partition 15, the second partition 21, and the pillar portions 20B, 20C, and the first substrate 30 opens to the bottom surface 10a. The flow path FA, the second flow path FB, and the third flow path FC are sealed. The first partition wall 15 has a first air storage section 19B, and the second partition wall 21 has a second air storage section 19C, so the strength is reduced. By joining to 30, the strength of the artificial multilayer tissue culture device 1 can be reinforced.

The observation device 2 is placed on the -Z side of the artificial multilayer tissue culture device 1. The observation device 2 is arranged at a position where the first communication path 16 and the second communication path 22 can be observed from the −Z side.

[Artificial multilayer tissue]
Next, an artificial multilayer tissue formed using the above-mentioned artificial multilayer tissue culture device 1 will be explained.
FIG. 5 is a partial plan view of an artificial multilayer tissue 50 cultured and formed using the artificial multilayer tissue culture device 1, viewed from the −Z side.

The artificial multilayer tissue 50 includes a dermal tissue layer (first tissue layer) 60 and an epidermal layer (second tissue layer) 70. The dermal tissue layer 60 is formed between the first flow path FA, the second opposing surface 18B in the first communication path 16, and between the opposing surface 18C in the second communication path 22. The dermal tissue layer 60 formed between the second opposing surfaces 18B and 18C of the opposing surfaces is formed on the second flow path FB side and the opposing surface 18C by interaction with the second opposing surface 18B and the opposing surface 18C. A curved meniscus is formed that bulges toward the 3-channel FC side.

The dermal tissue layer 60 is formed by culturing dermal cells (first cells) 62 in an extracellular matrix component 61. Examples of the extracellular matrix component 61 include, but are not limited to, 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) reconstituted basement membrane components, gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, thrombin, aprotinin, alginic acid, and the like.

As the dermal cells 62, 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 preferred. Human-derived fibroblasts include normal human dermal fibroblasts (NHDF), human pulmonary fibroblasts (HPF), human cardiac fibroblasts (HCF), and human aorta. These include adventitial fibroblasts: Human Aortic Adventitial Fibroblasts (HAoAF), human uterine fibroblasts: Human Uterine Fibroblasts (HUF), human villous mesenchymal fibroblasts: Human Villous Mesenchymal Fibroblasts (HVMF), etc. Fibroblast cells (NHDF) are preferred.

The epidermal layer 70 is formed by seeding and culturing epidermal cells (second cells) 71 together with a medium via the second channel FB on the dermal tissue layer 60 exposed to the first communication channel 16. It is. As the epidermal cells 71, 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 preferred. Human-derived epidermal cells include epidermal keratinocytes and epidermal melanocytes, with epidermal keratinocytes being preferred. Examples of epidermal keratinocytes include normal human epidermal keratinocytes (NHEK). Examples of epidermal melanocytes include normal human epidermal melanocytes (NHEM).

Note that the third channel FC may be a channel in which only a medium is supplied, or a channel in which a luminal tissue is formed by supplying a culture medium and luminal cells as third cells. good. Luminal cells include vascular cells. By culturing luminal cells in the third channel FC, a luminal layer can be formed on the wall surface of the third channel FC.

Examples of vascular cells include vascular epithelial cells and vascular endothelial cells, with vascular endothelial cells being preferred. Examples of vascular endothelial cells include cells derived from mammals such as humans, mice, and rats, and human-derived vascular endothelial cells are preferred. 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 Microvessels Endothelial cells (Human Dermal Microvascular Endothelial Cells: HDMEC), Human Dermal Blood Endothelial Cells (HDBEC), Human Dermal Lymphatic Endothelial Cells (HDLEC), Human Pulmonary Microvascular Endothelial Cells ( Human Pulmonary Microvascular Endothelial Cells (HPMEC), Human Cardiac Microvascular Endothelial Cells (HCMEC), Human Bladder Microvascular Endothelial Cells (HBdMEC), Human Uterine Microvascular Endothelial Cells (HBdMEC) Microvascular Endothelial Cells (HUtMEC), etc., and human umbilical vein endothelial cells (HUVEC) are preferred.

[Method for manufacturing artificial multilayer tissue 50]
Next, a method for manufacturing the artificial multilayer tissue 50 will be explained.
The method for manufacturing the artificial multilayer tissue 50 according to the present invention includes preparing the artificial multilayer tissue culture device 1, culturing dermal cells 62 in the first flow path FA to form the dermal tissue layer 60, and By supplying culture medium to the flow path FB and the third flow path FC, and adjusting the posture of the artificial multilayer tissue culture device 1, the epidermis is supplied to the second flow path FB located above the first flow path FA. The cells 71 are injected, the epidermal cells 71 are placed on the dermal tissue layer 60 exposed to the first communicating path 16, and the epidermal cells 71 placed on the dermal tissue layer 60 are cultured in the medium of the second flow path FB. including doing.

Hereinafter, a method for manufacturing the artificial multilayer tissue 50 will be described in detail with reference to FIGS. 6 to 10.

(Preparation of artificial multilayer tissue culture device 1)
In preparation for the artificial multilayer tissue culture device 1, the second substrate 30 is bonded to the lower surface 10a of the first substrate 10 using an adhesive or the like. This completes the preparation of the artificial multilayer tissue culture device 1.
When the second substrate 30 is bonded to the lower surface 10a of the first substrate 10, the first communicating path 16 has a rectangular cross section perpendicular to the Y-axis.

(Formation of dermal tissue layer 60)
When the preparation of the artificial multilayer tissue culture device 1 is completed, the dermal tissue layer 60 is formed. In forming the dermal tissue layer 60, first, a mixture (mixture) of the extracellular matrix component 61 and dermal cells 62 is injected into the first flow path FA from the inlet 11A shown in FIG. 2 under cooling. do. In order to smoothly inject the mixture from the inlet 11A into the first flow path FA, it is preferable to inject the artificial multilayer tissue culture device 1 in a posture in which the XY plane is parallel to the horizontal plane.

The extracellular matrix component 61 in this embodiment includes a mixture of multiple types of gels. Specifically, the extracellular matrix component 61 contains collagen (Atelocollagen; 1.0 mg/mL) and alginate (RGD-alginate; 5.0 mg/mL). Further, as the dermal cells 62, human dermal fibroblasts (NHDF; 5e5 cells/ml) were used.

When the mixture of the extracellular matrix component 61 and the dermal cells 62 is poured into the first flow path FA, the extracellular matrix component 61 containing the dermal cells 62 flows into the first communication path 16 as shown in FIG. A meniscus is formed that contacts the second facing surface 18B and swells toward the second flow path FB side. Similarly, the extracellular matrix component 61 including the dermal cells 62 contacts the opposing surface 18C in the first communicating path 16 to form a meniscus that bulges toward the third flow path FC.

Thereafter, the mixture is incubated under predetermined conditions while being kept moist. As an example, the culture was performed at a temperature of 37° C. for 60 minutes.

Collagen and alginate have different gelation conditions, and the above incubation causes collagen to gel first. After the collagen is gelled, calcium chloride (CaCl ₂ ) is added to the first channel FA from the inlet 11A. Calcium ions generated in the first channel FA by the addition of calcium chloride cause the alginate to gel. As a result, an extracellular matrix component 61 in which collagen and alginate are gelled is formed in the first channel FA.

Since alginate moves toward calcium ions, when calcium ions (calcium chloride) are added from the second flow path FB or the third flow path FC, the alginate moves toward the second flow path FB or the third flow path FC. There is a possibility that he will move to FC. Therefore, calcium chloride is preferably added from a position (for example, about 3 mm) away from the first communication path 16 and the second communication path 22 in the first flow path FA.

Here, since the gelled collagen is soft, when the epidermal cells 71 are seeded on the dermal tissue layer 60 exposed to the first communicating path 16, there is a possibility that the collagen will fall into the first flow path FA. On the other hand, in order to support the epidermal cells 71 without falling, it is possible to use a relatively hard gel such as fibrin, but fibrin is easily soluble, so even if the epidermal cells 71 are cultured, the epidermal layer 70 is not formed. fibrin may disappear.

On the other hand, alginate does not dissolve like fibrin and is harder than collagen, but has the property of being highly contractile. Therefore, in this embodiment, after the collagen solution and the alginate solution are supplied to the first flow path FA, the collagen is first gelled to form the base of the extracellular matrix. Then, the alginate is gelled in the presence of the collagen gel base. Even if large shrinkage occurs due to alginate gelation, the collagen gel base can alleviate the shrinkage.

Therefore, in this embodiment, by adding calcium ions to gel the alginate after the collagen has gelled, strength for supporting the seeded epidermal cells 71 can be secured and the extracellular matrix component 61 can be dissolved. It is compatible with restraint.

When the extracellular matrix component 61 is gelled in the first channel FA, as shown in FIG. inject. Examples of media MB and MC include keratinocyte media.

When medium MC is injected into the third flow path FC and medium MB containing epidermal cells 71 is injected into the second flow path FB, the posture of the artificial multilayer tissue culture device 1 is adjusted so that the second flow path FB becomes the first flow path. It is located above the flow path FA. That is, the artificial multilayer tissue culture device 1 is erected with the +Y side facing upward.

In the artificial multilayer tissue culture device 1 in which the +Y side is the upper side, the first portion 13B of the second flow path FB is horizontal, and the second portion 14B moves upward (+Y side) as it moves away from the first portion 13B. Lean. Therefore, the epidermal cells 71 located above the second portion 14B will settle in the medium MB due to their own weight, and then be guided to the inclined second portion 14B and accumulated in the first portion 13B. . Furthermore, since the wall surface facing the second flow path FB and the opposing surface 17B of the first partition wall 15 are subjected to a surface treatment to suppress adhesion of the epidermal cells 71, the epidermal cells 71 accumulated in the first portion 13B can be effectively accumulated and arranged on the dermal tissue layer 60 (extracellular matrix component 61) exposed to the first communication path 16, as shown in FIG.

Once the epidermal cells 71 are placed on the dermal tissue layer 60, they are incubated for, for example, one hour. As the epidermal cells 71 are cultured in the medium MB and the dermal cells 62 are cultured in the medium MC, as shown in FIG. is formed.

Oxygen is consumed when culturing dermal cells 62 and epidermal cells 71, but the first partition wall 15 is provided with a first air storage section 19B, the second partition wall 21 is provided with a second air storage section 19C, and the first partition wall 15. Since the PDMS forming the second partition wall 21 has air permeability, oxygen can be supplied to the first flow path FA, second flow path FB, and third flow path FC through air. . Therefore, the dermal tissue layer 60 and the epidermal layer 70 can be smoothly formed without causing oxygen deficiency.

Similarly, since the second substrate 30 is made of PDMS, oxygen can be supplied to the first flow path FA, the second flow path FB, and the third flow path FC via the second substrate 30.

These dermal tissue layer 60 and epidermal layer 70 are formed in the same plane parallel to the XY plane, and can be observed using the observation device 2 from the -Z side via the thin plate-shaped second substrate 30. can.

FIG. 9 shows a state in which the artificial multilayer tissue culture device 1 is erected with the +Y side facing upward, and 10-50 cells of epidermal cells 71 are arranged (seeded) on the dermal tissue layer 60 exposed to the first communicating path 16. This is an image captured.

FIG. 10 is an image of the dermal tissue layer 60 and the epidermal layer 70 in the first communicating path 16 after culturing the epidermal cells 71 for one day.
As shown in FIGS. 9 and 10, immediately after the epidermal cells 71 are placed on the dermal tissue layer 60, the epidermal cells 71 are removed from the dermis without falling into the first channel FA side and without the extracellular matrix component 61 disappearing. The formation of the epidermal layer 70 on the tissue layer 60 could be observed in the same sample.

FIG. 11 is a fluorescence image taken of the dermal tissue layer 60 and the epidermal layer 70 shown in FIGS. 9 and 10. FIG. 12 is a fluorescent image of the epidermis layer 70 taken in the Y direction. As shown in FIG. 11, in the epidermal layer 70 above the dermal tissue layer 60, it was observed that differentiation of a tissue with a thinly imaged nucleus occurred on the surface of the tissue with a clearly imaged nucleus. . Further, the skin layer 70 formed in the first communicating path 16 whose cross section perpendicular to the Y axis is rectangular is approximately rectangular when viewed in the Y direction, as shown in FIG. It was observed that they were formed to fill the space between them.

As explained above, in the artificial multilayer tissue culture device 1 of the present embodiment, the first flow path FA, the second flow path FB, and the third flow path FC are arranged in the same plane with grooves opening on the lower surface 10a. is formed on the first substrate 10, and the optically transparent second substrate 30 is bonded to the lower surface 10a, so that the artificial multilayer tissue 50 of the living body can be observed through the second substrate 30 without creating tissue sections. It becomes possible to do so.

Furthermore, in the artificial multilayer tissue culture device 1 of this embodiment, by continuing the culture by replacing the medium, it becomes possible to observe the passage of time with the same artificial multilayer tissue sample.

In addition, in the method for manufacturing an artificial multilayer tissue of this embodiment, collagen and alginate are used as the extracellular matrix component 61 for forming the dermal tissue layer 60, and the collagen is first gelled, and then the alginate is gelled. By doing so, it is possible to prevent the extracellular matrix component 61 from falling into the first channel FA or disappearing, and it becomes possible to form and observe the epidermal layer 70 in a normal state and position.

[Second embodiment of artificial multilayer tissue culture device]
Next, a second embodiment of the artificial multilayer tissue culture device will be described with reference to FIGS. 13 to 16.
In these figures, the same elements as those of the first embodiment shown in FIGS. 1 to 12 are denoted by the same reference numerals, and the explanation thereof will be omitted.
The second embodiment differs from the first embodiment described above in that an inlet for seeding epidermal cells is provided.

FIG. 13 is a schematic perspective view of an artificial multilayer tissue observation system SYS having an artificial multilayer tissue culture device 1 according to the second embodiment of the present invention. FIG. 14 is a plan view of the first substrate 10 of the second embodiment viewed from above. FIG. 15 is a partial plan view showing the manufacturing process of the artificial multilayer tissue 50. FIG. 16 is a partial plan view of the artificial multilayer tissue 50 cultured and formed using the artificial multilayer tissue culture device 1, viewed from the −Z side.

As shown in FIGS. 13 to 16, the first substrate 10 according to the second embodiment has an inlet 26 on the +Y side of the first communicating path 16 for seeding epidermal cells. The inlet 26 is disposed at a position where a portion thereof intersects with the second flow path FB, and penetrates the first substrate 10 in the Z direction. That is, the inlet 26 communicates with the second flow path FB. The length in the Y direction where the inlet 26 and the second flow path FB intersect is, for example, about 0.2 mm.
The other configurations are the same as those in the first embodiment.

In the artificial multilayer tissue culture device 1, when manufacturing the artificial multilayer tissue 50, after the extracellular matrix component 61 is gelled in the first flow path FA, as shown in FIG. Medium MC is injected into the third channel FC in a horizontal state, and medium MB containing epidermal cells 71 is injected from the inlet 26 into the second channel FB. At this time, since the inlet 26 communicates with the second flow path FB, a constant number of epidermal cells 71 can be injected.

Thereafter, the artificial multilayer tissue culture device 1 is erected with the +Y side facing upward. Thereby, the epidermal cells 71 injected into the second flow path FB from the inlet 26 settle in the medium MB due to their own weight, and are accumulated in the first portion 13B. At this time, since the wall surface facing the second flow path FB and the opposing surface 17B of the first partition wall 15 are subjected to surface treatment to suppress adhesion of the epidermal cells 71, the epidermal cells accumulated in the first portion 13B 71 can be effectively accumulated and arranged on the dermal tissue layer 60 (extracellular matrix component 61) exposed to the first communication path 16.

As described above, in the artificial multilayer tissue culture device 1 of the present embodiment, in addition to obtaining the same functions and effects as the artificial multilayer tissue culture device 1 of the first embodiment, the inlet 26 is connected to the second flow path. After injecting the epidermal cells 71 into the FB, the inlet 26 is emptied and filled with air, thereby creating an air-liquid interface 27 at the intersection of the inlet 26 and the medium MB, as shown in FIG. can be formed. Therefore, it becomes possible to culture the seeded epidermal cells 71 in the immediate vicinity of the air-liquid interface and promote differentiation of the epidermal layer 70. Furthermore, it is also possible to easily add fat-soluble reagents through this inlet 26.

Although 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 and combinations of the constituent members shown in the above example are merely examples, and can be variously changed based on design requirements and the like without departing from the gist of the present invention.

For example, in the embodiment described above, an artificial skin tissue is exemplified as an artificial multilayer tissue, but the invention is not limited to this as long as it is a layered tissue. The artificial multilayer tissue may be, for example, muscle tissue using skeletal muscle cells or cardiac muscle cells or lung tissue using lung cells instead of the above-mentioned fibroblasts. Furthermore, liver tissue using hepatocytes, intestinal tissue using intestinal cells, digestive system tissue such as pancreatic tissue using pancreatic cells, urinary system tissue such as kidney tissue using kidney cells, nervous system It may also be neural tissue using cells. When the present invention is applied to such tissues, the extracellular matrix used in the above embodiments does not necessarily need to be present, and may be one in which only various cells are accumulated.

Further, in the above embodiment, the artificial multilayer tissue culture device 1 is provided with one first communication path 16 and one second communication path 22, but the present invention is not limited to this configuration. A configuration in which the passage 16 and the second communication passage 22 are provided may be used. For example, a plurality of first communication passages 16 and second communication passages 22 are provided in the first portions 13B and 13C of the artificial multilayer tissue culture device 1 described in the first embodiment, and the epidermal cells 71 are grown in each of the first communication passages 16. They can be cultured under almost the same culture conditions. In this case, by observing the epidermal layer 70 formed in the plurality of first communication channels 16, it is possible to confirm the reproducibility of the epidermal layer 70 formed using one artificial multilayer tissue culture device 1. become. In the artificial multilayer tissue culture device 1 described in the first embodiment, in addition to the configuration in which the first portions 13B and 13C are provided with a plurality of first communication paths 16 and second communication paths 22, the first communication path 16 and the second communication path The configuration is such that a plurality of sets are provided in the flow path direction, each consisting of first portions 13B, 13C in which one passage 22 is provided, and second portions 14B, 14C on both sides of the first portions 13B, 13C. You can.

In addition, the first portions 13B and 13C in which the first communication path 16 and the second communication path 22 of the artificial multilayer tissue culture device 1 described in the second embodiment are provided, and the first portions 13B and 13C, respectively, are provided. A configuration may be adopted in which a plurality of sets each consisting of the second portions 14B, 14C on both sides and the inlet 26 provided near the first communication path 16 are provided in the flow path direction. In this configuration, similarly to the above, it is possible to confirm the reproducibility of the epidermal layer 70 formed using the same epidermal cells 71, or, for example, to confirm the reproducibility of the epidermal layer 70 formed using the same epidermal cells 71, and to Epidermal cells 71 for each organ can be seeded. When epidermal cells from multiple types of organs are seeded, it becomes possible to form and observe the epidermal layers 70 of different organs independently on the dermal tissue layer 60 under substantially the same conditions.

INDUSTRIAL APPLICATION This invention is applicable to the manufacturing method of an artificial multilayer tissue culture device and an artificial multilayer tissue.

DESCRIPTION OF SYMBOLS 1... Artificial multilayer tissue culture device, 10... First substrate, 10a... Lower surface (one side), 13B, 13C... First part, 14B, 14C... Second part, 15... First partition, 16... First communication path, 17B...Opposing surface, 18B...Second opposing surface, 19B...First air storage section, 19C...Second air storage section, 20B, 20C...Column section, 21...Second partition wall, 22...Second communication path, 26... Inlet, 30... Second substrate, 50... Artificial multilayer tissue, 60... Dermal tissue layer (first tissue layer), 62 Dermal cells (first cells), 70... Epidermal layer (second tissue layer), 71... Epidermal cells (second cell), FA...first channel, FB...second channel, FC...third channel

Claims (13)

a first channel that opens on one side of the first substrate and extends in the first direction, in which the first cells are cultured;
a second channel that is open on the one surface and extends in the first direction, and in which second cells are cultured;
a third channel that is open on the one surface, extends in the first direction, and is supplied with at least a culture medium;
a second substrate having a light transmittance that is bonded to the one surface and seals the first flow path, the second flow path, and the third flow path;
has
The second flow path is provided on one side of the first flow path in a second direction intersecting the first direction via a first partition,
The third flow path is provided on the other side in the second direction with respect to the first flow path via a second partition,
The first partition wall has a first communication path that communicates the first flow path and the second flow path,
The second partition wall has a second communication path that communicates the first flow path and the third flow path,
The first communication path has an opposing surface that faces the first direction and tapers from the second flow path toward the first flow path,
an inlet that penetrates the first substrate in a direction perpendicular to the one surface and communicates with the second flow path at a position above the opposing surface when the second flow path is located above the first flow path; An artificial multilayer tissue culture device characterized by being provided with . The first communication passage includes the opposing surface located on the second flow path side, and a second communication path located on the first flow path side that expands in diameter from a tapered end of the opposing surface toward the first flow path side. having two opposing surfaces;
The artificial multilayer tissue culture device according to claim 1. The second flow path is
a first portion arranged parallel to the first flow path and provided with the first communication path;
and second portions provided on both sides of the first portion in the first direction, the distance from the first flow path in the second direction increasing as the distance from the first portion in the first direction increases. ,
The artificial multilayer tissue culture device according to claim 1 or 2. A wall surface of the first partition facing the second flow path and a wall surface of the first communication path facing the second flow path are subjected to a surface treatment to suppress adhesion of the second cells.
An artificial multilayer tissue culture device according to any one of claims 1 to 3. The wall surface of the second partition facing the third flow path and the wall surface of the second communication path facing the third flow path are not subjected to the surface treatment.
The artificial multilayer tissue culture device according to claim 4. The first partition wall has a first air storage portion in a region separated from the first flow path, the second flow path, and the first communication path,
The second partition wall has a second air storage part in a region separated from the first flow path, the third flow path, and the second communication path,
The first substrate is made of polydimethylsiloxane.
An artificial multilayer tissue culture device according to any one of claims 1 to 5. The second substrate is made of polydimethylsiloxane.
The artificial multilayer tissue culture device according to claim 6. The first air accommodating part and the second air accommodating part are provided with column parts that extend in the normal direction of the one surface and whose tips are flush with the one surface.
The artificial multilayer tissue culture device according to claim 6 or 7. the first cell is a dermal cell,
the second cell is an epidermal cell,
An artificial multilayer tissue culture device according to any one of claims 1 to 8. a first channel that opens on one side of the first substrate and extends in the first direction, in which the first cells are cultured;
a second channel that is open on the one surface and extends in the first direction, and in which second cells are cultured;
a third channel that is open on the one surface, extends in the first direction, and is supplied with at least a culture medium;
a second substrate having a light transmittance that is bonded to the one surface and seals the first flow path, the second flow path, and the third flow path;
has
The second flow path is provided on one side of the first flow path in a second direction intersecting the first direction via a first partition,
The third flow path is provided on the other side in the second direction with respect to the first flow path via a second partition,
The first partition wall has a first communication path that communicates the first flow path and the second flow path,
The second partition wall has a second communication path that communicates the first flow path and the third flow path,
preparing an artificial multilayer tissue culture device in which the first communication path has opposing surfaces facing in the first direction and tapering from the second flow path toward the first flow path;
Cultivating first cells in the first channel to form a first tissue layer;
Supplying the second cells and a medium to the second flow path, and supplying the medium to the third flow path;
Adjust the posture of the artificial multilayer tissue culture device to position the second flow path above the first flow path, and place the second cells on the first tissue layer exposed to the first communication path. to place and
culturing the second cells disposed on the first tissue layer in the medium in the second channel;
A method for producing an artificial multilayer tissue, comprising: Forming the first tissue layer includes supplying a mixture of collagen, alginate, and first cells to the first channel;
culturing the mixed solution to gel the collagen;
adding calcium ions to the first channel after gelling the collagen to gel the alginate;
The method for producing an artificial multilayer tissue according to claim 10 . adding the calcium ions from a position away from the first communication path in the first flow path;
The method for producing an artificial multilayer tissue according to claim 11 . Supplying the second cells and the medium to the second flow path includes:
supplying the second cells and the medium from an inlet provided in communication with the second flow path on one side in the second direction from the first communication path;
The method for manufacturing an artificial multilayer tissue according to any one of claims 10 to 12 .

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