WO2017126532A1 - Tissu tridimensionnel artificiel et son procédé de fabrication, dispositif de perfusion pour le tissu tridimensionnel artificiel, et procédé d'évaluation de médicaments utilisant le tissu tridimensionnel artificiel - Google Patents

Tissu tridimensionnel artificiel et son procédé de fabrication, dispositif de perfusion pour le tissu tridimensionnel artificiel, et procédé d'évaluation de médicaments utilisant le tissu tridimensionnel artificiel Download PDF

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WO2017126532A1
WO2017126532A1 PCT/JP2017/001499 JP2017001499W WO2017126532A1 WO 2017126532 A1 WO2017126532 A1 WO 2017126532A1 JP 2017001499 W JP2017001499 W JP 2017001499W WO 2017126532 A1 WO2017126532 A1 WO 2017126532A1
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Prior art keywords
artificial
tissue
layer
dimensional tissue
dimensional
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English (en)
Japanese (ja)
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昌治 竹内
雄矢 森本
森　宣仁
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University of Tokyo NUC
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University of Tokyo NUC
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Priority to JP2017562836A priority Critical patent/JP6933855B2/ja
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  • the present invention relates to an artificial three-dimensional tissue and a manufacturing method thereof, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue.
  • Patent Document 1 discloses that a coated cell having a cell surface coated with a coating containing an extracellular matrix component is cultured to form a dermal tissue layer in which the coated cell is laminated, and an epidermal cell is formed on the dermal tissue layer.
  • a technique for manufacturing an artificial skin model by arranging and forming an epidermis layer is disclosed.
  • the present invention has been made in view of the above problems, and provides an artificial three-dimensional tissue having a high quality and a thick epidermal layer, a manufacturing method thereof, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue. For the purpose.
  • a method for producing an artificial three-dimensional tissue extending in a predetermined direction, a culture vessel having a culture space surrounded by side walls, and penetrating the opposing side walls into the culture space.
  • Preparing a device including a flow path forming member suspended along the predetermined direction, culturing a first cell in the culture space, and forming a first tissue layer through which the flow path forming member penetrates; Removing the flow channel forming member from the first tissue layer to form a perfusion channel that penetrates the first tissue layer; and allowing the culture medium to perfuse the perfusion channel while the first tissue layer is perfused.
  • the direction of applying at least one of the extension and compression is the direction in which the flow path forming member is suspended (perfusion flow path direction) or the first tissue layer and the second tissue layer are bent in the stacking direction.
  • the layer direction of the first tissue layer and the second tissue layer can be selected.
  • a configuration in which at least one of the extension and compression stimuli is applied along a straight line for example, bending of a biological model
  • the flow path forming member may be arranged along a curved surface to form a curved perfusion flow path, and at least one of stretching and compression may be applied along the curved line.
  • the first tissue layer and the second tissue layer are bent or curved, the first tissue layer and the second tissue layer are compressed to, for example, a layer closer to the bending center or the bending center.
  • the stimulus of extension is given to the layer on the far side.
  • the artificial three-dimensional tissue is manufactured by the manufacturing method of the first aspect of the present invention, the drug is brought into contact with the artificial three-dimensional tissue, and the drug is contacted.
  • a method for evaluating a drug using an artificial three-dimensional tissue comprising measuring the response of the artificial three-dimensional tissue to a stimulus or measuring the permeability of the drug to the artificial three-dimensional tissue.
  • an artificial three-dimensional tissue perfusion device in which perfusion is performed on an artificial three-dimensional tissue extending in a predetermined direction, the culture tank having a culture space surrounded by a side wall, and facing the culture tank
  • a support part that attaches and detachably supports a flow path forming member suspended along the predetermined direction in a region where the artificial three-dimensional tissue in the culture space is disposed through the side wall, and each of the opposing side walls.
  • An engagement portion provided to engage with the artificial three-dimensional tissue to suppress contraction of the artificial three-dimensional tissue in the predetermined direction, and to apply at least one stimulus of extension and compression to the artificial three-dimensional tissue
  • An artificial three-dimensional tissue perfusion device is provided that includes a stimulus imparting unit.
  • an artificial three-dimensional tissue extending in a predetermined direction, the perfusion channel extending through the inside and extending in the predetermined direction, and a buttock extending on the surface in a direction corresponding to the predetermined direction
  • an artificial three-dimensional tissue characterized by comprising:
  • an artificial three-dimensional tissue having a high quality and a thick epidermal layer a manufacturing method thereof, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue.
  • FIG. 1 is a perspective sectional view schematically showing an artificial skin tissue 1 according to an embodiment of the present invention.
  • 1 is a schematic configuration diagram of an artificial skin tissue manufacturing system 30.
  • FIG. 1 is an external perspective view of a first embodiment of a perfusion device 40.
  • FIG. 3 is an external perspective view of the perfusion device 40 with the illustration of the extension device 80 omitted. It is the front view which looked at the engaging part 42c in the axial direction.
  • FIG. 6 is a cross-sectional view taken along line AA in FIG. 5. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue.
  • FIG. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a figure which shows the manufacture procedure of the artificial skin tissue. It is a photograph figure which shows the result at the time of giving the irritation
  • FIG. 3 is a cross-sectional view taken along a vertical plane including a length direction of a biological model F.
  • FIG. It is a figure which shows the manufacture procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacture procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacture procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacture procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacture procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment. It is a figure which shows the manufacture procedure of the artificial skin tissue 1 which concerns on 2nd Embodiment.
  • an artificial three-dimensional tissue and a manufacturing method thereof, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue of the present invention will be described with reference to FIGS.
  • an example in which an artificial skin tissue is manufactured as an artificial three-dimensional tissue will be described.
  • FIG. 1 is a perspective sectional view schematically showing an artificial skin tissue 1 which is an artificial three-dimensional tissue.
  • the artificial skin tissue 1 includes a dermis tissue layer (first tissue layer) 10 and an epidermis layer (second tissue layer) 20.
  • the artificial skin tissue 1 has a predetermined direction (horizontal direction in FIG. 1) along a plane orthogonal to the direction in which the dermis tissue layer 10 and the epidermis layer 20 are laminated (vertical direction in FIG. 1, hereinafter referred to as a lamination direction). Hereinafter, it is formed to extend in the first direction).
  • the epidermis layer 20 is formed by seeding and culturing epidermis cells (second cells) 21 on the dermis tissue layer 10.
  • epidermis cells for example, epidermal keratinocytes can be used.
  • 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.
  • epidermal keratinocytes include normal human epidermal keratinocytes (NHEK).
  • epidermal melanocytes include normal human epidermal melanocytes (NHEM).
  • the dermal tissue layer 10 is formed by culturing dermal cells (first cells) 12 in the extracellular matrix component 11.
  • the extracellular matrix component 11 is not particularly limited.
  • collagen type I, type II, type III, type V, type XI, etc.
  • mouse EHS tumor extract type IV collagen, laminin, heparan sulfate proteoglycan, etc.
  • a base membrane component trade name: Matrigel
  • gelatin agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, etc.
  • fibroblasts can be used as the dermal cells 12, for example.
  • fibroblasts include cells derived from mammals such as humans, mice and rats, and human-derived fibroblasts are preferred.
  • Human-derived fibroblasts include human skin fibroblasts (Normal Human Dermal Fibroblasts: NHDF), human lung fibroblasts: Human Pulmonary Fibroblasts (HPF), human cardiac fibroblasts: Human Cardiac Fibroblasts (HCF), human aorta Outer membrane fibroblasts: Human Aortic vent Adventitial Fibroblasts (HAoAF), human uterine fibroblasts: Human Uterine Fibroblasts (HUF), human chorionic mesenchymal fibroblasts: Human Villous Mesenchymal Fibroblasts (HVMF) Fibroblasts (NHDF) are preferred.
  • the dermis tissue layer 10 has a perfusion channel 13 that extends through the inside of the dermis tissue layer 10 in the first direction.
  • the perfusion channel 13 is a channel through which a medium (details will be described later) is perfused when the epidermal cells 21 are cultured.
  • a lumen layer 15 formed using vascular cells 14 is provided on the surface of the perfusion channel 13.
  • the vascular cells 14 for example, endothelial cells can be used.
  • vascular cells examples include vascular epithelial cells and vascular endothelial cells, and vascular endothelial cells are preferred.
  • vascular endothelial cells examples 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 (Human Umbilical Vein Endothelial Cells: HUVEC), human umbilical artery endothelial cells (Human Umbilical Artery Endothelial Cells: HUAEC), human coronary artery endothelial cells (Human Coronary Artery Cellous: HCAEC), human saphenous vein endothelial cells (Human Saphenous Vein Endothelial Cells: HSaVEC), human human pulmonary artery endothelial cells (Human Pulmonary Artery Endothelial Cells: HPAEC), human human aortic endothelial cells (Human Aortic ⁇ Endothelial Cells: HAoEC) Endothelial cells (Human Dermal Endothelial : Cells: HDMEC), Human skin vascular endothelial cells (Human Dermal H Blood Endothelial Cells: HDBEC), Human skin
  • FIG. 2 is a schematic configuration diagram of the artificial skin tissue manufacturing apparatus 30.
  • the artificial skin tissue manufacturing apparatus 30 includes a perfusion device (artificial three-dimensional tissue perfusion device) 40, a culture dish 50, and a pump 60.
  • a perfusion device artificial three-dimensional tissue perfusion device
  • the pump 60 supplies the culture medium to the perfusion device 40 via the pipe 61.
  • the pump 60 collects the medium M discharged to the culture space 45 via the perfusion device 40 via the pipe 62.
  • FIG. 3 is an external perspective view of the first embodiment of the perfusion device 40.
  • the perfusion device 40 includes a culture tank 41, a connector (support part) 42, a wire (a linear member, a flow path forming member) 43, and an extension device 80. As shown in FIG. 2, the culture tank 41 is disposed inside the culture dish 50.
  • FIG. 4 is an external perspective view of the perfusion device 40 with the extension device 80 omitted.
  • the culture tank 41 is formed in a rectangular tube shape having a culture space 45 surrounded by the side wall 44 and penetrating in the vertical direction.
  • the side wall 44 is provided in a rectangular shape in plan view.
  • the culture tank 44 is made of a flexible material such as silicon rubber.
  • the lower end of the culture space 45 can be switched between closed and opened by a bottom plate 47 (see FIG. 7).
  • the bottom plate 47 is detachably provided between the opposing side walls 44a and 44b.
  • the culture space 45 is opened at the lower end by removing the bottom plate 47 and closed at the lower end by attaching the bottom plate 47.
  • the connector 42 has a mounting portion (cylinder portion) 42a, a connecting portion 42b, an engaging portion 42c, and a through hole 42d penetrating therethrough.
  • the mounting portion 42 a is formed in a shaft shape and is mounted through the side wall 44 of the culture tank 41.
  • the connection part 42b is provided at one end of the mounting part 42b.
  • the connection part 42b is arrange
  • the engaging portion 42c is provided at the other end of the mounting portion 42b.
  • the engaging part 42 c is arranged in the culture space 45 of the culture tank 41 with a gap from the side wall 44.
  • FIG. 5 is a front view of the engaging portion 42c viewed in the axial direction.
  • 6 is a cross-sectional view taken along line AA in FIG.
  • the engaging portion 42 c is spaced from the outer peripheral surface of the mounting portion 42 a by a second cylinder portion 42 e that is arranged coaxially with a gap in the circumferential direction of the mounting portion 42 a.
  • a plurality of arranged rib portions 42f are examples of rib portions 42f.
  • the rib portion 42f connects the outer peripheral surface of the mounting portion 42a and the inner peripheral surface of the second cylindrical portion 42e.
  • Four rib portions 42f are provided at intervals of 90 degrees.
  • a gap 42g surrounded by the mounting portion 42a, the second cylindrical portion 42e, and the rib portion 42f penetrates the engaging portion 42c in the axial direction.
  • a plurality of pairs (six pairs in FIG. 4) of the connectors 42 are mounted on the opposing side walls 44 at positions where the through holes 42d are coaxial.
  • the three pairs of connectors 42 are arranged along the first direction, and the other three pairs of connectors 42 are arranged along a second direction orthogonal to the first direction in the horizontal direction.
  • a lyophilic process for the extracellular matrix component 11 As the lyophilic treatment, for example, O 2 plasma treatment can be adopted. This lyophilic treatment also serves as a treatment for improving cell adhesion.
  • the wire 43 is a linear member used to form the perfusion channel 13.
  • the wire 43 is supported so as to be attached to and detached from a connector 42 that is coaxially mounted on the opposite side wall 44.
  • the wire 43 is inserted (supported) into the through-hole 42d of the connector 42 and can be suspended in a region where the dermal tissue layer 10 of the culture space 45 is disposed.
  • the wire 43 is formed of, for example, a polyamide resin.
  • the extension device 80 includes sandwiching portions 81 and 82, a crank portion 83, a rotation motor 84, and a base 85.
  • the base 85 is integrally fixed to the upper part of the culture dish 50.
  • the sandwiching portion 81 is integrally provided so as to sandwich one side wall 44a from both sides in the thickness direction of the pair of side walls 44 facing each other.
  • the sandwiching portion 81 sandwiches the side wall 44a over the entire height direction.
  • the sandwiching portion 81 has an opening 81a in a region where the connector 42 is disposed. Since the opening 81 a is formed in the sandwiching portion 81, the connector 42 is prevented from interfering with the sandwiching portion 81.
  • the clamping part 81 is fixed to the base 85 at the upper end.
  • the sandwiching portion 82 is integrally provided so as to sandwich the side wall 44b facing the side wall 44a from both sides in the thickness direction, out of the pair of side walls 44 facing each other.
  • the sandwiching portion 82 sandwiches the side wall 44b over the entire height direction.
  • the clamping part 82 has an opening 82a in a region where the connector 42 is disposed. Since the opening 82 a is formed in the holding portion 82, the connector 42 is prevented from interfering with the holding portion 82.
  • the clamping part 82 is connected to the crank part 83 at the upper end.
  • the crank portion 83 is connected to the clamping portion 82 at one end in the length direction so as to be rotatable (oscillated) around an axis extending in the vertical direction.
  • the crank portion 83 is integrated with the rotating portion 84a that rotates integrally with the rotary motor 84 fixed to the base 85 on the other end side in the length direction in the length direction at a position eccentric to the rotation center.
  • the extension device 80 can reciprocate in the length direction according to the position in the length direction of the connecting portion with the rotation portion 84a when the rotation motor 84 rotates.
  • the crank part 83 moves in the length direction
  • the holding part 82 connected to the crank part 83 and holding the side wall 44b moves in the length direction.
  • the clamping portion 81 that clamps the side wall 44 a is fixed to the base 85. Therefore, the culture tank 41 formed of a flexible material is elastically deformed in a direction in which the side walls 44a and 44b face each other (hereinafter simply referred to as a facing direction) in accordance with the movement of the holding portion 82, so that the side wall 44b Move in the opposite direction.
  • the side wall 44b moves in the facing direction
  • the distance between the connectors 42 provided facing the side walls 44a and 44b increases or decreases.
  • a method for manufacturing an artificial skin tissue (artificial three-dimensional tissue) 1 is a method for manufacturing an artificial three-dimensional tissue 1 extending in a predetermined direction, and includes a culture tank 41 having a culture space 45 surrounded by a side wall 44, and A perfusion device (artificial three-dimensional tissue perfusion device, device) 40 including a wire (linear member, flow path forming member) 43 suspended through the opposing side wall 44 in the culture space 45 in a predetermined direction.
  • Preparing culturing the dermis cells 12 in the extracellular matrix component 11 in the culture space 45 to form the dermis tissue layer 10 through which the wire 43 penetrates, and removing the wire 43 from the dermis tissue layer 10 to dermis Forming the perfusion channel 13 penetrating the tissue layer 10 and culturing the epidermis cells 21 arranged on the dermis tissue layer 10 while perfusing the perfusion channel 13 to form the epidermis layer 20. , At least in culture of epidermal cells 21, and applying at least one of the stimulation of the compression and extension along a predetermined direction in the dermal tissue layer 10 and skin layer 20, a.
  • the manufacturing method of the artificial skin tissue 1 will be described in detail. 7 to 15, only the perfusion device 40 is illustrated as appropriate, and the culture dish 50, the pump 60, and the like are not illustrated.
  • the illustration of the holding portions 81 and 82 in the extension device 80 is omitted as appropriate, and the bottom plate 47 is illustrated and attached to or removed from the side wall 44 in order to facilitate understanding. explain.
  • the perfusion device 40 is prepared by mounting the connector 42 so that the through-hole 42 d is coaxial with the opposing side wall 44 of the culture tank 41, and the bottom plate 47 at the lower end of the side wall 44. Is attached to close the lower end side opening of the culture space 45.
  • the wire 43 is inserted into the through-hole 42 d that is coaxial, and the wire 43 is suspended in the culture space 45.
  • the lyophilic treatment is performed on at least an area of the connector 42 exposed to the culture space 45.
  • the lyophilic treatment may be performed on the connector 42 before being mounted on the culture tank 41 or may be performed on the connector 42 mounted on the side wall 44.
  • the surface of the culture tank 41 facing the culture space 45 is coated with a sealing material.
  • a sealing material a material that does not adversely affect the extracellular matrix component 11, dermal cells 12, and epidermal cells 21, for example, polyparaxylylene (hereinafter referred to as parylene) is formed by a film forming method such as vapor deposition.
  • the film thickness of the sealing material is preferably 1/100 to 1/10 with respect to the gap of the mounting portion and the gap of the mounting portion.
  • the film thickness of the sealing material is preferably 1/50 to 1/10, more preferably 1/50 to 1/20 with respect to the gap of the mounting portion and the gap of the mounting portion.
  • the sealing material is formed with a film thickness (1/25) of 2 ⁇ m with respect to a gap of about 50 ⁇ m.
  • the dermal tissue layer 10 is formed.
  • a mixture of the extracellular matrix component 11 and the dermis cells 12 is poured into the culture space 45 of the culture tank 41.
  • the mixture is poured in an amount that makes the wire 43 soaked in height.
  • collagen is used as the extracellular matrix component 11.
  • the mixture of the extracellular matrix component 11 and the dermal cells 12 is poured into the culture tank 41, the mixture is cultured (incubated) under predetermined conditions.
  • the culture condition is such that the density of the dermis tissue layer 10 is equivalent to that of human dermis.
  • the culture conditions were 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 is engaged with the engaging portion 42c, the shrinkage in the stacking direction is not restricted, but the shrinkage in the first direction and the second direction is restricted. More specifically, as shown in FIG. 6, the extracellular matrix component 11 is engaged with the second cylindrical portion 42e and the rib portion 42f of the engaging portion 42c from the side opposite to the center of the culture space 45. Therefore, the second cylinder part 42e and the rib part 42f serve as a barrier and the contraction toward the center side is suppressed.
  • the extracellular matrix component 11 is crimped
  • the engaging portion 42c is lyophilicized with respect to the extracellular matrix component 11, the extracellular matrix component 11 is in close contact with the engaging portion 42c with a greater adhesion force in the first direction. And the resistance force with respect to the shrinkage
  • the stacking direction is contracted, the contraction in the first direction and the second direction is suppressed, and the interior is connected to the wire 43.
  • a dermal tissue layer 10 is formed.
  • epidermis cells 21 are seeded on the dermis tissue layer 10.
  • the epidermal cells 21 are cultured while the medium M is perfused from the pump 60 through the pipe 61 and the through-hole 42d to the perfusion channel 13.
  • the epidermal cells 21 are cultured by gas-liquid culture in which the medium M in the perfusion channel 13 is diffused through the dermis tissue layer 10 while exposing the epidermal cells 21 to gas.
  • the epidermal cells 21 can be induced to differentiate to form the epidermal layer 20.
  • the culture conditions for forming the skin layer 20 were 9 days at a temperature of 37 ° C.
  • the medium M perfused into the perfusion channel 13 through one connector 42 is discharged into the inner space of the culture dish 50 through the other connector 42 and the pipe 63.
  • a part of the medium M diffused from the perfusion channel 13 to the dermis tissue layer 10 is discharged into the internal space of the culture dish 50 through the opening 46 a and the groove 46 c of the culture tank 41.
  • the medium M discharged to the culture dish 50 is collected via the pipe 62.
  • the supply amount of the culture medium M from the pump 60 and the recovery amount of the culture medium M through the pipe 62 are such that the lower side of the dermis tissue layer 10 is immersed in the culture medium M when the epidermal layer 20 is cultured, and the epidermal cells 21 become air.
  • the liquid level of the medium M in the culture dish 50 is set so as to be exposed.
  • the rotation motor 84 in the extension device 80 rotates and the crank portion 83, the clamping portion 82, and the side wall 44b move in the length direction, so that the skin layer 20 is provided on the side wall 44b.
  • the connector 42 moves in a direction away from or approaching the connector 42 provided on the fixed side wall 44a.
  • the connecting portion between the crank portion 83 and the rotating portion 84a moves away from the side wall 44a when the rotary motor 84 rotates
  • the connector 42 provided on the side wall 44b is fixed as shown in FIG.
  • the dermis tissue layer 10 and the epidermis layer 20 supported by the connector 42 are given stimulation for extension.
  • the above-described stimulation of extension to the dermis tissue layer 10 and epidermis layer 20 and stimulation of compression (or release from extension) are applied at a predetermined frequency.
  • a predetermined frequency 0.03 Hz or more and 0.25 Hz or less are preferable, for example.
  • the frequency is lower than the lower limit, the period becomes longer and the effect of applying the stimulus may not be sufficiently obtained. If the frequency exceeds the upper limit, the cycle may be shortened and the skin layer 20 having a predetermined thickness may not be obtained.
  • the amount to be stretched or compressed with respect to the dermis tissue layer 10 and the epidermis layer 20 is preferably a strain amount of ⁇ 20% with respect to the distance between the connectors 42 provided on the side walls 44a and 44b, and a strain amount of ⁇ 10%. Is more preferable.
  • the present invention relates to a method for evaluating skin irritation of a drug using the artificial skin tissue 1 of the present invention.
  • the drug in the present invention includes drugs such as pharmaceuticals, cosmetics and quasi drugs. 1
  • the evaluation method of the present invention for example, the drug can be evaluated in an environment close to the actual skin as compared with the conventional method.
  • the evaluation method of the present invention is extremely useful, for example, in the evaluation of the dynamics of drugs of various molecular weights in the creation (screening) of new drugs, and in the development of cosmetics and quasi drugs.
  • the evaluation method of the present invention can be performed, for example, by bringing a drug into contact with the artificial skin tissue and measuring a response to a stimulus due to the contact of the drug.
  • the response can be measured, for example, by measuring transcutaneous electrical resistance.
  • medical agent means the substance used as evaluation object, for example, an inorganic compound, an organic compound, etc. are mentioned.
  • FIGS. 16A and 16B are photographic views showing a cross section of the artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 by the manufacturing method described above.
  • FIG. 16A is a photographic diagram showing the results when the above-described stretching stimulus is applied at 0.1 Hz and the strain amount is ⁇ 5%.
  • FIG. 16B is a photographic diagram showing the result when the above-described extension stimulus is not applied.
  • FIG. 17 (a), (b), and (c) are photographic views showing a cross section of the artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 in a state where the medium M is not perfused.
  • FIG. 17 (a) is a photographic diagram showing the results when applying a stretching stimulus of 0.06 Hz and a strain amount of ⁇ 10%.
  • FIG. 17B is a photographic diagram showing the results when the extension stimulus is 0.1 Hz and the strain amount is ⁇ 10%.
  • FIG. 17 (c) is a photographic diagram showing the results when the extension stimulus is 0.2 Hz and the strain amount is ⁇ 10%.
  • FIG. 18A, 18B, and 18C are fluorescence images showing a cross section of the artificial skin tissue 1 in which the epidermis layer 20 is cultured on the dermis tissue layer 10 in a state where the medium M is not perfused.
  • FIG. 18 (a) is a fluorescence image showing the results when the extension stimulus is applied at 0.06 Hz and the strain amount is ⁇ 10%.
  • FIG. 18B is a fluorescence image showing the results when the extension stimulus is 0.1 Hz and the strain amount is ⁇ 10%.
  • FIG. 18C is a fluorescence image showing the results when the extension stimulus is 0.2 Hz and the strain amount is ⁇ 10%.
  • the basal portion 20 a formed of keratin 15 (Keratin 15) is cultured on the dermis tissue layer 10 side of the epidermis layer 20. It was observed that the surface layer portion 20b formed of keratin 10 was cultured on the surface side.
  • the repulsive force was measured as robustness. As shown in FIG. 19, the repulsive force is measured by using a force gauge FG that supports the back side of the artificial skin tissue 1 supported by the perfusion device 40 and compresses the epidermis layer 20. The repulsive force when compressed was measured.
  • FIG. 20 shows the repulsion when the skin layer formed by applying the above-described extension stimulus and the skin layer formed without applying the extension stimulus are compressed by the force gauge FG to cause a displacement of 300 ⁇ m. It is the result of measuring force.
  • FIG. 21 shows the repulsion when the skin layer formed by applying the above-described extension stimulus and the skin layer formed without applying the extension stimulus are compressed by the force gauge FG to cause a displacement of 500 ⁇ m. It is the result of measuring force.
  • the skin layer formed by applying an extension stimulus was measured to have a repulsion force twice or more that of the skin layer formed without applying an extension stimulus at a displacement of 300 ⁇ m.
  • FIG. 20 shows the repulsion when the skin layer formed by applying the above-described extension stimulus and the skin layer formed without applying the extension stimulus are compressed by the force gauge FG to cause a displacement of 300 ⁇ m.
  • the skin layer formed by applying an extension stimulus measures about 1.6 times the repulsive force at a displacement of 500 ⁇ m than the skin layer formed without applying an extension stimulus. It was done. In any case, it was confirmed that when the epidermal layer was formed by applying an extension stimulus, it had greater robustness than when no extension stimulus was applied.
  • FIG. 22 shows the epidermis in the artificial skin tissue 1 in which the epidermal layer 20 is cultured on the dermis tissue layer 10 by applying a stimulation of extension at 0.2 Hz and a strain amount of ⁇ 10% in a state where the medium M is not perfused.
  • 3 is a photograph showing the surface state of a layer 20.
  • FIG. 22 it was confirmed that the elongate portion extending in the extending direction and the direction orthogonal to the extending direction was formed on the surface of the skin layer 20 formed by the manufacturing method of the present embodiment. Therefore, the artificial skin tissue 1 according to the present embodiment has the epidermis layer 20 closer to the human body.
  • FIG. 23 is a diagram schematically showing a mechanism for measuring the percutaneous absorption characteristics of the artificial skin tissue 1 using the perfusion device 40.
  • a reservoir 49 is placed on the epidermis layer 20 in the artificial skin tissue 1.
  • the solution L containing caffeine and ISDN is used for measuring the transdermal absorption characteristics.
  • the solution L contains 9.4 ⁇ mol of caffeine and 3.8 ⁇ mol of ISDN. This solution L is stored in the reservoir 49.
  • the percutaneous absorption characteristics of the artificial skin tissue 1 permeate the artificial skin tissue 1 from the reservoir 49 and reach the lower side of the artificial skin tissue 1, and the artificial skin tissue 1 and the perfusion channel 13 from the reservoir 49.
  • the amounts of caffeine and ISDN were measured for the medium MB that reached the through-hole 42d of the connector 42 by perfusion.
  • the endothelium functionality of the perfusion channel 13 it measured in each state of the presence or absence of a vascular endothelial growth factor.
  • FIG. 24 is a diagram showing the results of measuring the percutaneous absorption characteristics of the artificial skin tissue 1, with the elapsed time from the start of perfusion as the horizontal axis, and the amount of caffeine or ISDN based on the initial amounts of the media MA and MB.
  • the amount of substance is shown as the vertical axis.
  • FIG. 24A shows the relationship between the amount of caffeine substance measured in the medium MA and time.
  • FIG. 24B shows the relationship between the amount of ISDN substance measured in the medium MA and time.
  • FIG. 24C shows the relationship between the amount of caffeine substance measured in the medium MB and time.
  • FIG. 24D shows the relationship between the amount of ISDN substance measured in the medium MB and time.
  • the relationship between the presence or absence of vascular endothelial growth factor is shown.
  • FIGS. 1 to 24 the same components as those of the first embodiment shown in FIGS. 1 to 24 are denoted by the same reference numerals, and the description thereof is omitted or simplified.
  • the planar artificial skin tissue 1 is manufactured is illustrated in the first embodiment, the second embodiment will be described using an example in which the artificial skin tissue 1 having a curved cross-sectional shape is manufactured.
  • FIG. 25 is a schematic perspective view of a perfusion device 40A in which a biological model F is provided in the culture tank 41.
  • the fabric model F is a model imitating a finger.
  • FIG. 26 is a cross-sectional view taken along a vertical plane including the length direction (predetermined direction, first direction) of the biological model F.
  • 27 to 31 are cross-sectional views taken along a plane orthogonal to the length direction of the biological model F.
  • the biological model F is provided to be exposed to the culture space 45 from the bottom wall 46 of the culture tank 41 with the dorsal surface as a curved surface portion 70.
  • the joint between the bottom wall 46 of the culture tank 41 and the biological model F is a material that does not adversely affect the extracellular matrix component 11, the dermal cells 12, and the epidermal cells 21, for example, polyparaxylylene (hereinafter referred to as parylene). ) Is deposited by a deposition method such as vapor deposition and sealed.
  • the wire 43 is suspended so that the fingertip side is lowered according to the inclination of the curved surface portion 70 with respect to the first direction.
  • a plurality of wires 43 (five wires in FIG.
  • each wire 43 and the biological model F are provided at intervals in the circumferential direction of the biological model F.
  • the distance between each wire 43 and the biological model F is set to a distance at which the thickness of the dermis tissue layer 10 formed between the wire 43 and the biological model F can secure a predetermined value.
  • the number of the wires 43 is appropriately determined according to the width (length in the circumferential direction) of the skin layer 20.
  • the mixture of the extracellular matrix component 11 and the dermal cells 12 When the mixture of the extracellular matrix component 11 and the dermal cells 12 is poured into the culture tank 41, the mixture is cultured under predetermined conditions. The cultured mixture of the extracellular matrix component 11 and the dermal cells 12 is restrained from contracting in the direction in which the wire 43 is suspended, and contracts in the other direction. Therefore, as shown in FIG. A dermal tissue layer 10 having a shape along the line is formed.
  • a plurality (five in this embodiment) of perfusion channels 13 are formed in the dermis tissue layer 10 as shown in FIG.
  • a vascular layer 15 is formed by injecting (seeding) vascular cells 14 onto the surface of the dermis tissue layer 10 facing the perfusion channel 13 and culturing for a certain period of time.
  • epidermal cells 21 are seeded on the dermis tissue layer 10.
  • the epidermal cells 21 are gas-liquid cultured while the medium M is perfused through the perfusion channel 13.
  • the clamping part 82 that clamps the side wall 44b moves in a direction away from or approaching the side wall 44a, so that the dermis tissue layer 10 and the epidermis layer 20 supported by the connector 42 are moved.
  • a compression stimulus is applied from the stretched state.
  • the epidermal cells 21 can be induced to differentiate to form the epidermal layer 20.
  • the artificial skin tissue 1 having a curved shape in which the dermis tissue layer 10 and the epidermis layer 20 are along the curved surface portion 70 is obtained.
  • an artificial skin tissue 1 having a thick epidermis layer 20 is obtained as compared with the case where no extension stimulus is applied.
  • a thick epidermis along the curved surface portion 70 of the biological model F is obtained.
  • a high-quality artificial skin tissue 1 having the layer 20 can be easily manufactured. Therefore, in the present embodiment, by preparing and using the biological model F of the desired site, the high-quality artificial skin tissue 1 having the thick epidermis layer 20 in the shape of the desired site can be easily obtained.
  • an artificial skin provided with a perfusion channel 13 having a repelling force closer to the human body and having a repulsive force closer to the human body and having a repelling force that extends in the direction of extension and the direction orthogonal to the direction of extension.
  • the tissue 1 can be easily obtained.
  • the finger skin tissue is exemplified as the artificial skin tissue 1.
  • the present invention can also be applied when artificially manufacturing the skin tissue of another part. is there.
  • an artificial skin tissue is exemplified as the artificial three-dimensional tissue.
  • the present invention is not limited to this configuration, and the present invention can be applied to other artificial tissues.
  • the handle O 2 plasma treatment as lyophilic engaging section 42c is not limited to this, it may take the other lyophilic treatment.
  • an adhesive for skin may be used instead of the lyophilic treatment.
  • a skin adhesive it is preferable to select a material that does not adversely affect the extracellular matrix component 11 and the dermal cells 12.
  • the structure which uses the wire 43 which is a linear member as a flow-path formation member for forming the perfusion flow path 13 produces with a gel material or a polymer other than the wire 43, for example.
  • a configuration using 3 fibers with or without cells may be used.
  • the perfusion channel 13 is not limited to a linear configuration.
  • planar flow path forming member for example, a mesh material can be used.
  • the configuration in which the stimulation of stretching / compression is applied to the dermis tissue layer 10 and the epidermis layer 20 in the direction in which the wire 43 or the perfusion channel 13 extends is exemplified, but the configuration is not limited thereto.
  • the configuration is not limited thereto.
  • FIG. Furthermore, it is good also as a structure which gives the irritation
  • compression stimulation can be applied to the layer near the bending center or bending center in the dermis tissue layer 10 and the epidermal layer 20, and extension stimulation can be applied to the far layer.
  • the configuration in which the perfusion channel 13 is used to perfuse the medium for culturing the epidermis cells 21 to form the epidermis layer 20 is not limited to this configuration.
  • a configuration may be used in which a drug is applied to the tissue surface (for example, the epidermis layer 20) and the perfusion channel 13 is used to evaluate the uptake of the drug into the perfusion channel 13. Further, the drug is mixed into the medium flowing through the perfusion channel 13, and the perfusion channel 13 is used to evaluate the diffusion of the drug from the perfusion channel 13 to the dermal tissue layer 10 or the epidermis layer 20. There may be.
  • the perfusion channel 13 When the perfusion channel 13 is used in such a configuration, it is not necessary to form the perfusion channel 13 before the formation of the epidermis layer 20, and the procedure for forming the perfusion channel 13 after the formation of the epidermis layer 20 is performed. There may be. In addition, when the evaluation target is, for example, only the dermis tissue layer 10, it is not necessary to form the epidermis layer 20.
  • the artificial skin tissue 1 is exemplified as the artificial three-dimensional tissue, but is not limited to this.
  • the artificial three-dimensional tissue may be, for example, a muscle tissue using skeletal muscle cells or cardiomyocytes instead of the fibroblasts described above.
  • it is a digestive system tissue such as a liver tissue using hepatocytes, a pancreas tissue using pancreatic cells, a urinary system tissue such as a kidney tissue using kidney cells, and a nerve tissue using neural cells. Also good.
  • the extracellular matrix used in the above embodiment does not necessarily exist, and may be a collection of only various cells.
  • a high-quality artificial three-dimensional tissue and a manufacturing method thereof, an artificial three-dimensional tissue perfusion device, and a drug evaluation method using the artificial three-dimensional tissue can be provided.
  • this invention is useful in fields, such as cosmetics, a pharmaceutical, a pharmaceutical, etc., for example.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne un procédé de fabrication de tissu tridimensionnel artificiel, qui comprend les étapes suivantes : préparer un dispositif de perfusion pourvu d'une cuve de culture possédant un espace de culture entouré par des parois latérales, et un composant formant un circuit fluidique pénétrant au travers des parois latérales opposées et suspendu selon une direction prédéterminée dans l'espace de culture ; cultiver un premier groupe de cellules dans l'espace de culture et former une première couche de tissu à travers laquelle pénètre le composant formant un circuit fluidique ; retirer le composant formant un circuit fluidique de la première couche de tissu et installer un circuit fluidique de perfusion traversant la première couche de tissu ; cultiver un second groupe de cellules au-dessus de la première couche de tissu tout en apportant par perfusion un milieu de culture à travers le circuit fluidique de perfusion, et former une seconde couche de tissu ; et appliquer une stimulation d'extension et/ou une stimulation de compression à la première couche de tissu et à la seconde couche de tissu, au moins pendant la culture du second groupe de cellules.
PCT/JP2017/001499 2016-01-20 2017-01-18 Tissu tridimensionnel artificiel et son procédé de fabrication, dispositif de perfusion pour le tissu tridimensionnel artificiel, et procédé d'évaluation de médicaments utilisant le tissu tridimensionnel artificiel Ceased WO2017126532A1 (fr)

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JPWO2024071436A1 (fr) * 2022-09-28 2024-04-04
WO2025053290A1 (fr) * 2023-09-07 2025-03-13 国立大学法人佐賀大学 Procédé de renforcement de la résistance d'un corps tissulaire tridimensionnel

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JP2019122335A (ja) * 2018-01-18 2019-07-25 国立大学法人 東京大学 人工三次元組織のバリア機能測定システム、人工三次元組織のバリア機能測定方法及び人工三次元組織を用いた薬剤評価方法
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JPWO2024071436A1 (fr) * 2022-09-28 2024-04-04
WO2024071436A1 (fr) * 2022-09-28 2024-04-04 国立大学法人佐賀大学 Corps de tissu tridimensionnel et son procédé de production
WO2025053290A1 (fr) * 2023-09-07 2025-03-13 国立大学法人佐賀大学 Procédé de renforcement de la résistance d'un corps tissulaire tridimensionnel

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