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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
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  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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  • C12M23/00—Constructional details, e.g. recesses, hinges
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/069—Vascular Endothelial cells
  • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
  • G01N15/0806—Details, e.g. sample holders, mounting samples for testing
• • G—PHYSICS
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5064—Endothelial cells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1347—Smooth muscle cells
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1352—Mesenchymal stem cells
  • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/28—Vascular endothelial cells
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
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  • C12N2535/00—Supports or coatings for cell culture characterised by topography
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
  • G01N2015/086—Investigating permeability, pore-volume, or surface area of porous materials of films, membranes or pellicules
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N2015/1006—Investigating individual particles for cytology

Definitions

• the present disclosure relates to a blood vessel model.
• microchannels which are channels with micrometer order widths.
• the pores in the porous membrane used in conventional internal organ models are produced using what is known as a track etching process, in which, for example, a material configuring the porous membrane is irradiated with heavy ions. Accordingly, the opening coverage ratio of pores in the membrane is, for example, as low as from 2% to 20%, and due to the membrane also being thick, the passage of red blood cells or the like is obstructed by the porous membrane. Namely, in the conventional internal organ models, there were cases in which the level of drug induced injury to a layer of cells provided at a surface of the porous membrane may not be accurately evaluated.
• a blood vessel model that may enable movement of red blood cells or the like to be suppressed from being obstructed by a porous membrane during an extravasation test.
• a blood vessel model includes: a pair of channel members, mutually opposing each other, each of which includes an opposing face in which a respective microchannel is formed; and a porous membrane that includes plural through-holes penetrating in a thickness direction, that is disposed between the opposing faces of the pair of channel members, and that partitions between the microchannels, wherein the porous membrane is provided with a vascular endothelial cell layer so as to cover one face facing one of the microchannels, an average opening diameter of the through-holes is from 1 ⁇ to 20 ⁇ , and an opening coverage ratio of the through-holes is from 30% to 70%.
• the average opening diameter of the through-holes in the porous membrane partitioning between the microchannels is from 1 ⁇ to 20 ⁇ , and the opening coverage ratio of the through-holes is from 30% to 70%.
• a membrane thickness of the porous membrane may be less than or equal to half of the average opening diameter of the through-holes.
• the membrane thickness of the porous membrane is less than or equal to half of the average opening diameter of the through-holes, compared to a case in which the membrane thickness of the porous membrane is greater than half the average opening diameter of the openings of the through-holes, red blood cells or the like may further readily pass through the through-holes in the porous membrane. Accordingly, the second aspect may further improve the accuracy of the extravasation test.
• communication holes that place the through-holes in communication with each other may be formed inside the porous membrane; the through-holes may be arranged in a honeycomb pattern; a variation coefficient of opening diameters of the through-holes may be less than or equal to 10%; and a porosity of the porous membrane may be greater than or equal to 50%.
• the through-holes are arranged in a honeycomb pattern and are in communication with each other through the communication holes.
• the variation coefficient of the opening diameters of the openings of the through-holes is less than or equal to 10%), and the porosity of the porous membrane is greater than or equal to 50%.
• the red blood cells or the like may be made to pass through more uniformly. Accordingly, the third aspect may further improve the accuracy of the extravasation test.
• a cell layer of cells may be selected from the group consisting of smooth muscle cells,
• mesenchymal stem cells may be provided at the other face of the porous membrane facing the other microchannel.
• a blood vessel model that more closely resembles an actual blood vessel may be achieved.
• a tensile elongation at break of the porous membrane may be greater than or equal to 50%; and a stress required for 10% elongation of the porous membrane may be less than or equal to 1000 gf/mm 2 .
• the porous membrane is formed from a flexible material having a tensile elongation at break greater than or equal to 50% and having a stress required for 10% elongation less than or equal to 1000 gf/mm 2 , a blood vessel model that more closely resembles an actual blood vessel may be achieved.
• the through-holes may have flattened shapes in plan view and may include a major axis and a minor axis.
• the through-holes have flattened shapes such as elliptical shapes in plan view, the red blood cells or the like may pass more readily through the through-holes. Accordingly, the sixth aspect may further improve accuracy of the extravasation test.
• the porous membrane may include a porous region in which the through-holes are formed, and a non-porous region in which the through-holes are not formed.
• the seventh aspect since, for example, portions of the porous membrane disposed in the vicinity of inlets and in the vicinity of outlets of the microchannels are configured as the non-porous regions in which the through-holes are not formed, the flow of red blood cells or the like inside the microchannels may be regulated. Accordingly, the seventh aspect may further improve accuracy of the extravasation test.
• the present disclosure may enable the movement of red blood cells or the like during extravasation testing to be suppressed from being obstructed by the porous membrane.
• Fig. 1 is a perspective diagram illustrating an overall configuration of a blood vessel model of an exemplary embodiment
• Fig. 2 is an exploded perspective diagram illustrating overall configuration of a blood vessel model of an exemplary embodiment
• Fig. 3 is an enlarged cross-section illustrating a porous membrane of a blood vessel model of an exemplary embodiment
• Fig. 4 is a plan view illustrating a porous membrane of a blood vessel model of an exemplary embodiment
• Fig. 5 is a plan view illustrating a porous membrane of a blood vessel model of a modified example
• Fig. 6 is a plan view illustrating a porous membrane of a blood vessel model of a modified example
• Fig. 7A is a micrograph of a porous membrane of an example 1;
• Fig. 7B is a micrograph of a porous membrane of a comparative example 1;
• Fig. 8A is a result of image fluorescence in the microchannels of the
• Fig. 8B is a result of image fluorescence in the microchannels of the
• Fig. 9A is a result of a FITC-Dextran permeability test in the cell-layer-affixed blood vessel models of an example 3;
• Fig. 9B is a result of a FITC-Dextran permeability test in the cell-layer-affixed blood vessel models of a comparative example 3;
• Fig. 1 OA is a micrograph of the porous membrane of an example 4.
• Fig. 1 OB is a partial enlarged view of Fig. 10A.
• Fig. 11 is a micrograph of the porous membrane of an example 5.
• a blood vessel model 10 of an exemplary embodiment includes an upper channel member 12 and a lower channel member 14 stacked on one another.
• the upper channel member 12 and the lower channel member 14 are, for example, configured from an elastic material such as poly dimethyl siloxane (PDMS), and have substantially rectangular plate shapes.
• PDMS poly dimethyl siloxane
• cyclic olefin polymer COP
• epoxy resin urethane resin
• styrene-based thermoplastic elastomer olefin-based thermoplastic elastomer
• acrylic-based thermoplastic elastomer polyvinyl alcohol and the like.
• a concave portion 18, that defines an upper microchannel 16 is formed in a lower face of the upper channel member 12, namely, at an opposing face 12A opposing to the lower channel member 14.
• the concave portion 18 includes an inlet 18A, an outlet 18B, and a channel portion 18C communicating the inlet 18A with the outlet 18B.
• Through-holes 20 A and 20B are formed in the upper channel member 12, penetrate the upper channel member 12 in the thickness direction, and have lower ends in respective communication with the inlet 18A and the outlet 18B. Upper ends of the through-holes 20A and 20B open at an upper face of the upper channel member 12. Liquid supply tubing (not illustrated) is connected to the upper ends of the through-holes 20A and 20B.
• a concave portion 24 that defines a lower microchannel 22 is formed in an upper face of the lower channel member 14, namely, at an opposing face 14A opposing to the upper channel member 12.
• the concave portion 24 includes an inlet 24A, an outlet 24B, and a channel portion 24C communicating the inlet 24A with the outlet 24B.
• the inlet 24 A and outlet 24B of the lower channel member 14 and the inlet 18 A and outlet 18B of the upper channel member 12 are provided at positions not overlapping in plan view.
• the channel portion 24C of the lower channel member 14 and the channel portion 18C of the upper channel member 12 are provided at positions overlapping in plan view.
• Through-holes 26A and 26B are also formed in the upper channel member 12, penetrate the upper channel member 12 in the thickness direction, and have lower ends in respective communication with the inlet 24 A and the outlet 24B. Upper ends of the through-holes 26A and 26B open at the upper face of the upper channel member 12. Liquid supply tubing (not illustrated) is connected to the upper ends of the through-holes 26A and 26B.
• a porous membrane 28 is provided between the opposing faces 12A and 14A of the upper channel member 12 and the lower channel member 14.
• the upper channel member 12 and the lower channel member 14 are joined together with the porous membrane 28 in an interposed state therebetween.
• a variety of methods such as welding, attraction (self-adhering), or joining with bolts, may be employed as the method for joining the upper channel member 12 and the lower channel member 14 together.
• the porous membrane 28 is, for example, a hydrophobic polymer that solves in a hydrophobic organic solvent.
• the hydrophobic organic solvent is a liquid with a solubility in 25°C water of less than or equal to 10 (g/lOOg water).
• hydrophobic polymers include polymers such as polybutadiene, polystyrene, polycarbonate, polyesters (for example, polylactic acid, polycaprolactone, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polylactic
• acid-polycaprolactone copolymer polyethylene terephthalate, polyethylene naphthalate, polyethylene succinate, polybutylene succinate, and poly-3-hydroxybutyrate
• polyacrylate polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyhexafluoropropene, polyvinyl ether,
• polyvinylcarbazole polyvinyl acetate, polytetrafluoroethylene, polylactone, polyamide, polyimide, polyurethane, polyurea, polyaromatics, polysulfone, polyethersulfone,
• polysiloxane derivatives for example, triacethyl cellulose, cellulose acetate propionate, and cellulose acetate butyrate.
• cellulose acylate for example, triacethyl cellulose, cellulose acetate propionate, and cellulose acetate butyrate.
• Polymers that dissolve in a hydrophobic organic solvent are preferable from the viewpoint of producing a honeycomb membrane using the production method disclosed, for example, in JP-B No. 4,734, 157.
• These polymers may have the form of a homopolymer, a copolymer, a polymer blend or a polymer alloy, as necessary, from the viewpoints of, for example, solubility in solvents, optical properties, electrical properties, membrane strength, and elasticity. These polymers may be used singly, or in combination of two or more thereof.
• the material of the porous membrane 28 is not limited to being a hydrophobic polymer, and various materials may be selected from viewpoints such as the adhesiveness of cells.
• An upper face 28A and a lower face 28B of the porous membrane 28 (hereinafter, the upper face 28A and the lower face 28B are may be referred collectively as "main faces") are sized so as to substantially cover the channel portions 18C and 24C of the upper microchannel 16 and the lower microchannel 22, such that the upper microchannel 16 is partitioned from the lower microchannel 22.
• the upper face 28 A of the porous membrane 28 namely, the main face facing the upper channel member 12, together with the concave portion 18 of the upper channel member 12, defines the upper microchannel 16.
• a vascular endothelial cell layer 36 is provided to the upper face 28A of the porous membrane 28 so as to completely cover the upper face 28 A.
• the inside of the upper microchannel 16 thereby configures an environment that closely resembles the inside of a blood vessel.
• vascular endothelial cells include: vascular endothelial cells originating from the umbilical vein, the umbilical artery, the aorta, a coronary artery, the pulmonary artery, a pulmonary microvessel, or a dermal microvascular; and vascular endothelial cells differentiated from pluripotent stem cells.
• a cell layer 38 for example, configured from cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblast cells, is provided to the lower face 28B of the porous membrane 28 so as to completely cover the lower face 28B.
• the lower microchannel 22 thereby configures an environment that closely resembles a blood vessel exterior.
• Mesenchymal stem cells are somatic stem cells that are capable of dividing into muscle cells, fat cells, cartilage cells, and the like.
• the cell layer 38 of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblast cells may be provided to the upper face 28 A of the porous membrane 28, and the vascular endothelial cell layer 36 may be provided to the lower face 28B of the porous membrane 28. Moreover, it is sufficient that the vascular endothelial cell layer 36 be provided to at least one of the main faces of the porous membrane 28. Configuration may be such that the cell layer 38 is not provided to the other main face of the porous membrane 28.
• a region where cells are seeded on at least one of the upper face 28A and the lower face 28B of the porous membrane 28 is coated by at least one selected from the group consisting of fibronectin, collagen (for example, type I collagen, type IV collagen or type V collagen), laminin, vitronectin, gelatin, perlecan, nidogen, proteoglycan, osteopontin, tenascin, nephronectin, a basement membrane matrix and polylysine.
• vascular endothelial cell layer 36 and the cell layer 38 For providing the vascular endothelial cell layer 36 and the cell layer 38 to the respective main face of the porous membrane 28, for example, a method in which a cell suspension is poured into the upper microchannel 16 and the lower microchannel 22 so as to seed cells on the main faces of the porous membrane 28, may be employed. Further, a method in which cells are seeded and cultured on the main faces of the porous membrane 28 inside a separate culturing apparatus, and then the porous membrane 28 having the vascular endothelial cell layer 36 and the cell layer 38 formed thereon is mounted in the blood vessel model 10, may also be employed.
• plural through-holes 30 are formed in the porous membrane 28 penetrating the porous membrane 28 in the thickness direction. Openings 30A of the through-holes 30 are provided in each of the upper face 28 A and the lower face 28B of the porous membrane 28. As illustrated in Fig. 4, the openings 30A have circular shapes in plan view. The openings 30A are provided at a separation from each other. A flat portion 32 extends between adjacent openings 30A. Note that the openings 30A are not limited to circular shapes, and may also be configured with polygonal shapes.
• the plural openings 30A are arranged in a regular manner, and in the present exemplary embodiment, as illustrated in Fig. 4, for example, are arranged in honeycomb pattern.
• a honeycomb pattern arrangement is an arrangement in which the centers of the openings 30A are disposed at positions of the vertexes, and at the points where diagonals intersect, for units of a parallel hexagon (a regular hexagon is preferable) or a shape close to this.
• centers of the openings means the centers of the openings 30A in plan view.
• the arrangement of the openings 30A is not limited to a honeycomb pattern.
• the openings 30A may also be configured in a lattice pattern or a face-centered lattice pattern.
• a lattice pattern arrangement is an arrangement in which the centers of the openings are disposed at the positions of the vertexes for units of a parallelogram (it goes without saying that this includes squares, rectangles, and rhombuses; a square is preferable) or a shape close thereto.
• a face-centered lattice pattern arrangement is an arrangement in which the centers of the openings are disposed at the positions of the vertexes, and at the points where diagonals intersect, for units of a parallelogram (it goes without saying that this includes squares, rectangles, and rhombuses; a square is preferable) or a shape close thereto.
• the arrangement of the openings 30A may be arbitrary. However, it is preferable that the plural openings 30A are arranged in a regular manner from the viewpoint of achieving a uniform density of the openings 30A in the upper face 28 A and the lower face 28B of the porous membrane 28.
• a regular arrangement is an arrangement in which the variation coefficient of the surface areas of the parallel hexagon or parallelogram units of the arrangement is, for example, less than or equal to 10%. Some of the openings 30A may be missing or the openings 30A may be not in alignment. However, it is preferable that the openings 30A are continuously arranged in all directions without any gaps therebetween.
• the "variation coefficient" is a value arrived at by dividing the standard deviation of a given population by the mean thereof, and is an index expressing the degree of dispersion in the population as a percentage.
• each through-hole 30 in the porous membrane 28 has a spherical segment shape, which is a shape in which the upper and lower end of a sphere have been cut away.
• Through -holes 30 that are adjacent to one another are in communication with each other through respective communication holes 34 in the interior of the porous membrane 28.
• each through-hole 30 is in communication with every adjacent through-hole 30.
• each through-hole 30 is in respective communication with six adjacent through-holes 30 through six
• the through-holes 30 may have a barrel shape, a circular column shape, a polygonal column shape, or the like, and the communication holes 34 may be tube shaped voids linking together adjacent through-holes 30.
• An opening diameter D of each opening 30A of the through-holes 30 is, for example, a size such that red blood cells in blood are able to pass therethrough.
• the average opening diameter is preferably from 1 ⁇ to 20 ⁇ , and even more preferably is from 3 ⁇ to 10 ⁇ . Setting the average opening diameter to 1 ⁇ or greater enables the through-holes 30 to be a size that allows red blood cells to pass therethrough, and setting the average opening diameter to 20 ⁇ or less enables retention of the vascular endothelial cell layer 36 and the cell layer 38 on the main faces of the porous membrane 28.
• opening diameter D is the major axis of the openings 30A
• average opening diameter is the calculated average of the opening diameters D measured for ten or more arbitrarily selected openings 30A.
• Major axis means the longest distance between two arbitrarily selected points on the outline of an opening. However, in cases in which a direction has been specified, “major axis” means the longest distance between two arbitrarily selected points along that direction.
• the opening coverage ratio of the openings 30A of the through-holes 30 is preferably from 30% to 70%, and even more preferably is from 40% to 60%. Setting the opening coverage ratio to greater than or equal to 30% enables the movement of red blood cell to be suppressed from being obstructed by the porous membrane 28, and setting the opening coverage ratio to less than or equal to 70% enables the required strength to be achieved in the porous membrane 28.
• opening coverage ratio expresses a ratio of S2 to SI as a percentage, wherein SI denotes a unit of surface area of the porous membrane 28 under the supposition that the main faces of the porous membrane 28 are smooth (namely, under the supposition that there are no openings 30A in the porous membrane 28), and S2 denotes the sum of the surface area of openings 30A provided per unit surface area, where the same units of measurement are used for SI and S2.
• the membrane thickness T of the porous membrane 28 is preferably less than or equal to half of the average opening diameter of the openings 30A of the through-holes 30. Specifically, the thickness T is preferably from 0.5 ⁇ to 10 ⁇ , and is more preferably from 1 ⁇ to 10 ⁇ . Setting the membrane thickness T of the porous membrane 28 to a thickness less than or equal to half of the average opening diameter of the through-holes 30 enables the movement of red blood cell to be suppressed from being obstructed by the porous membrane 28.
• the porous membrane 28 is a scaffold upon which cells adhere and grow
• cell-cell interaction between the cells on one face of the porous membrane 28 and the cells on the other face of the porous membrane 28, namely, at least one of information transmission via humoral factors or cell-cell contact becomes more active the greater the opening coverage ratio on the porous membrane 28 and the thinner the membrane thickness of the porous membrane 28.
• the more active cell-cell interaction is during cell cultivation to provide the vascular endothelial cell layer 36 and the cell layer 38 on the main faces of the porous membrane 28, the better a blood vessel model can be produced with functionality resembling that of in vivo tissue.
• the variation coefficient of the opening diameters D of the openings 30A is preferably less than or equal to 10%, and the smaller the variation coefficient the more preferable. The smaller the variation coefficient of the opening diameters D, the more uniformly red blood cells and the like can pass through the plural through-holes 30 in the porous membrane 28.
• the porosity of the porous membrane 28 is preferably greater than or equal to 50%. Setting the porosity to greater than or equal to 50% enables the movement of red blood cell to be suppressed from being obstructed by the porous membrane 28. Note that if the porosity is too large, the strength of the porous membrane 28 becomes insufficient with regards to the required strength therefor, and so the porosity is preferably less than or equal to 95%.
• porosity expresses a ratio of V2 to VI as a percentage, wherein VI denotes a unit of volume of the porous membrane 28 under the supposition that the main faces of the porous membrane 28 are smooth (namely, under the supposition that there are no openings 30A in the porous membrane 28), and V2 denotes the sum of the volume of the through-holes 30 and the communication holes 34 provided per unit volume, where the same units of measurement are used for VI and V2.
• the tensile elongation at break of the porous membrane 28 is preferably greater than or equal to 50%, is more preferably 100%, and is even more preferably greater than or equal to 200%.
• the stress required for 10% elongation of the porous membrane 28 is preferably less than or equal to 1000 gf/mm 2 .
• a material becomes more flexible as the tensile elongation at break increases and the stress required for 10% elongation decreases. It is thus possible to bend, stretch, and compress the porous membrane 28, enabling the blood vessel model 10 to more closely resemble an actual blood vessel.
• the "tensile elongation at break” can be evaluated by measuring the elongation at tensile breaking of the porous membrane 28 according to the method defined in JIS K 6251 :2010.
• the "stress required for 10% elongation” can be evaluated by measuring the stress applied to the porous membrane 28 when the porous membrane 28 is elongated by 10% according to the method defined in JIS K 6251 :2010.
• examples of methods for producing the porous membrane 28 formed with the through-holes 30 include, nano-printing processes, condensation processes, etching processes, sandblasting processes, or press molding processes.
• a nano-printing process is a method in which the through-holes 30 are produced by pouring a material for configuring the porous membrane 28 into a mold having projections and recesses, or pressing such a mold against a material for configuring the porous membrane 28.
• a condensation process is a method in which condensation is induced on the surface of a material for configuring the porous membrane 28, so as to form the through-holes 30 by using water droplets as molds.
• a condensation process enables the membrane thickness of the porous membrane 28 to be made thinner, enables the porosity and the opening coverage ratio of the openings 30A to be increased, and also enables the
• the porous membrane 28 is produced using a condensation process. Condensation processes are described in detail in, for example, JP-B No. 4945281, JP-B No. 5422230, JP-B No. 5405374, and Japanese Patent Application Laid-Open (JP-A) No. 2011-74140. [0060] Next, as an example, explanation is given regarding a case in which a drug toxicology evaluation is performed using the blood vessel model 10 of the present exemplary embodiment.
• the upper channel member 12 and the lower channel member 14 are joined together with the porous membrane 28 in an interposed state therebetween to produce the blood vessel model 10 including the upper microchannel 16 and the lower microchannel 22, as illustrated in Fig. 2.
• the vascular endothelial cell layer 36 and the cell layer 38 are provided to the main faces of the porous membrane 28.
• a blood dilution containing a drug is run through tubing (not illustrated in the drawings) and the through-hole 20A into the upper microchannel 16, is passed through the inside of the upper microchannel 16, and is caused to pass through the through-hole 20B and tubing (not illustrated in the drawings) to run out of the blood vessel model 10.
• a culture solution or a physiological saline solution is run through tubing (not illustrated in the drawings) and the through-hole 26A into the lower microchannel 22, is passed through the inside of the lower microchannel 22, and is caused to pass through the through-hole 26B and tubing (not illustrated in the drawings) to run out of the blood vessel model 10.
• the pressure in the upper microchannel 16 through which the blood dilution flows is higher than that in the lower microchannel 22 through which the culture solution or the physiological saline solution flows.
• the vascular endothelial cell layer 36 is injured by the toxicity of the drug.
• the cell layer 38 is also injured by the drug.
• the state of the cells configuring the cell layer 38 changes due to cell-cell interaction between the vascular endothelial cell layer 36 and the cell layer 38, and as a result, a gap may be formed in the cell layer 38.
• a drug and a solution containing a tracer may be run through the upper microchannel 16 in place of the blood dilution.
• the tracer include fluorescently-labeled chemical substances, radioisotope-containing chemical substances, dye compounds, and the like, and more specifically, at least one selected from the group consisting of dextran, Evans blue, fluorescein sodium, and FITC microbeads.
• fluorescent dye is preferably red, with an excitation wavelength / fluorescence wavelength of 580 nm/605 nm.
• the level of drug induced injury to the vascular endothelial cell layer 36 and the cell layer 38 can be evaluated by measuring fluorescent intensity, radiation, or chromaticity in accordance with the type of tracer so as to quantify the tracer, and measuring the amount of tracer that has passed through the porous membrane 28 and flowed into the lower
• microchannel 22 from the upper microchannel 16.
• the present exemplary embodiment is configured such that in the porous membrane 28 partitioning the upper microchannel 16 from the lower microchannel 22, the average opening diameter of the openings 30A of the through-holes 30 is from 1 ⁇ to 20 ⁇ , and the opening coverage ratio of the openings 30A of the through-holes 30 is from 30% to 70%. Accordingly, during extravasation testing when red blood cells running through the upper microchannel 16 pass through the through-holes 30 in the porous membrane 28 and move into the lower microchannel 22, the movement of red blood cells can be suppressed from being obstructed by the porous membrane 28.
• the present exemplary embodiment is configured such that the membrane thickness of the porous membrane 28 is less than or equal to half of the average opening diameter of the openings 30A of the through-holes 30. Accordingly, compared to a case in which the membrane thickness of the porous membrane 28 is greater than half the average opening diameter of the openings 30A of the through-holes 30, red blood cells more readily pass through the through-holes 30 in the porous membrane 28. Accordingly, the present exemplary embodiment may further improve the accuracy of the extravasation test. [0071] Additionally, the present exemplary embodiment is configured with the openings 30A of the through-holes 30 arranged in a honeycomb pattern, and through-holes 30 within the porous membrane 28 are in communication with each other through the communication holes 34.
• the variation coefficient of the opening diameters of the openings 30A of the through-holes 30 is less than or equal to 10%, and the porosity of the porous membrane 28 is greater than or equal to 50%. Accordingly, red blood cells can pass through the plural through-holes 30 in the porous membrane 28 more uniformly. Accordingly, the present exemplary embodiment may further improve the accuracy of the extravasation test.
• the present exemplary embodiment is configured with the vascular endothelial cell layer 36 provided to the upper face 28 A of the porous membrane 28, and with the cell layer 38 provided to the lower face 28B of the porous membrane 28.
• the cell layer 38 is configured of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblast cells.
• the porous membrane 28 is configured from a flexible material having a tensile elongation at break that is greater than or equal to 50%, and in which the stress required for 10% elongation is less than or equal to 1000 gf/mm 2 .
• the blood vessel model 10 may be configured to more closely resemble an actual blood vessel.
• openings 30A of the through-holes 30 in the porous membrane 28 of the above exemplary embodiment have circular shapes in plan view
• openings 50A of through-holes 50 in a porous membrane 48 may have elliptical shapes in plan view, as illustrated in Fig. 5.
• the openings 50A of the through-holes 50 with elliptical shapes, for example, disc-shaped red blood cells may be easily passed through the openings 50A of the through-holes 50, and other cells in blood may be less liable to pass therethrough.
• Examples of methods for forming the openings 50A of the openings 50A of through-holes 50 into elliptical shapes include a method in which, after circular shaped openings 30A such as illustrated in Fig. 4 have been formed in the porous membrane 48, the porous membrane 48 is stretched along one direction (the left-right direction in Fig. 4). This method enables plural elliptical shaped openings 50Ato be formed having their major axis directions along the same direction (the left-right direction in Fig. 5).
• elliptical shaped openings 50A may be directly formed in the porous membrane 48 using press molding or the like, without stretching the porous membrane 48. Moreover, so long as the shape of the openings 50A is a flattened shape with a major axis and a minor axis in plan view, the shape of the openings 50A may, for example, be a flattened polygonal shape arrived at by stretching a regular polygon.
• the openings 30A of the through-holes 30 were arranged in a regular manner over the entirety of the main faces of the porous membrane 28.
• a porous membrane 58 may be provided with a porous region 62 in which openings 60A of through-holes 60 are formed, and a non-porous region 64 not formed with the openings 60A of through-holes 60 (the region marked by the double-dotted dashed line in Fig. 6).
• portions disposed in the vicinity of the inlet 18A and in the vicinity of the outlet 18B of the concave portion 18 configuring the upper microchannel 16 illustrated in Fig. 1, and in the vicinity of the inlet 24 A and in the vicinity of the outlet 24B of the concave portion 24 configuring the lower microchannel 22 illustrated in Fig. 1, are, for example, configured as the non-porous region 64.
• the flow of liquid such as blood is easily disturbed at the inlets 18A and 24 A and at the outlets 18B and 24B.
• the porous membrane 58 in the vicinity of the inlets 18A and 24A and in the vicinity of the outlets 18B and 24B as the non-porous region 64, the flow of liquid such as blood in the upper microchannel 16 and in the lower microchannel 22 may be regulated. Accordingly, the porous membrane 58 may further improve the accuracy of the extravasation test.
• the blood vessel model of the present disclosure may enable extravasation testing to be performed, in a state in which movement of leaking substances such as red blood cells to outside the blood vessel accompanying drug toxicity is suppressed from being obstructed by the porous membrane.
• the blood vessel model of the present disclosure may therefore be useful as a blood vessel model capable of toxicology testing with high-accuracy.
• FIG. 7 A illustrates a micrograph of a porous membrane of example 1.
• a porous membrane formed of polycarbonate similar to the porous membrane 28 of the above exemplary embodiment, was employed in which the openings of plural through-holes were arranged in a honeycomb pattern and the through-holes were in communication through communication holes.
• the average opening diameter of openings in the porous membrane of example 1 was 5 ⁇
• the opening coverage ratio of the openings was 55%
• the membrane thickness of the porous membrane was 2.2 ⁇
• the variation coefficient of the opening diameters of the openings was 3.5%
• the porosity of the porous membrane was 75%
• the tensile elongation at break was 250%
• the stress required for 10% elongation was 100 gf/mm2.
• microstructure of the produced porous membrane was measured using a profile scanning laser microscope (product name VK-X100, made by Keyence, Japan).
• the average opening diameter and the opening coverage ratio were achieved by performing binarization processing and image processing on the micrograph, using the 2D image analysis software WinROOF (Mitani Corp.).
• the membrane thickness of the porous membrane is an average value of the thickness of opening portions measured at ten points using the profile scanning laser microscope.
• RT-2002D-D (made by Rheotech Corp.).
• Fig. 7B illustrates a micrograph of a porous membrane of comparative example 1.
• a porous membrane of conventional technology formed of polycarbonate was employed in which the openings were formed by a track etching process.
• the average opening diameter of the openings in the porous membrane of comparative example 1 was 5.7 ⁇
• the opening coverage ratio of the openings was 12.4%
• the membrane thickness of the porous membrane was 10.6 ⁇
• the variation coefficient of the opening diameters of the openings was 35%
• the porosity of the porous membrane was 15%
• the tensile elongation at break was 150%
• the stress required for 10% elongation was 5800 gf/mm 2 .
• a porous membrane is prepared with medical paper affixed to both sides thereof.
• the medical paper on one face of the porous membrane is removed using tweezers, and the face from which the medical paper has been removed is set face down on a lower channel member.
• the porous membrane is then soaked in ethanol using a swab to join the porous membrane and the lower channel member together.
• the medical paper on the other face of the porous membrane is removed using tweezers, and the upper channel member is stacked on the other face of the porous membrane.
• the positions of the upper channel member and the lower channel member are aligned while being checked on a microscope, and the upper channel member and the lower channel member are joined together.
• the blood vessel model of example 1 and the blood vessel model of the comparative example 1 were respectively produced.
• the porous membranes employed did not have a vascular endothelial cell layer 36 nor a cell layer of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblast cells provided on principal faces thereof.
• a blood vessel model with a cell layer affixed thereto was produced by taking the blood vessel model of example 1 and forming a layer of rat vascular endothelial cells on the upper face of the porous membrane and forming a layer of cells composed of rat smooth muscle cells on the lower face of the porous membrane.
• a blood vessel model with a cell layer affixed thereto was produced by taking the blood vessel model of comparative example 1 and forming a layer of rat vascular endothelial cells on the upper face of the porous membrane and forming a layer of cells composed of rat smooth muscle cells on the lower face of the porous membrane.
• Rat Arterial Endothelial Cells produced by Angio-Proteomie were employed for the rat vascular endothelial cells
• Rat Aortic Smooth Muscle Cells produced by Lonza were employed for the rat smooth muscle cells.
• the lower microchannel was initially seeded with 100 [iL of a cell suspension of rat smooth muscle cells having a cell concentration of 3 ⁇ 10 6 cells/ml. After a day of cultivation, the upper microchannel was seeded with 100 ⁇ _, of a cell suspension of rat vascular endothelial cells having a cell concentration of 3 ⁇ 10 6 cells/ml. After two days of cultivation, the
• the internal pressure of the lower microchannel was set to approximately 1.3 kPa so as to establish parameters close to the blood flow and blood pressure conditions inside actual blood vessels.
• comparative example 1 both had permeability to red blood cells under conditions equivalent to those of blood pressure. Further, in comparison to the porous membrane of comparative example 1, the porous membrane of example 1 was more readily permeable to red blood cells, enabling confirmation that the porous membrane of the present exemplary embodiment enables the movement of red blood cells to be suppressed from being obstructed.
• the fluorescent beads had a diameter of 4 ⁇ and were labeled with red fluorescent dye with an excitation wavelength of 580 nm and a fluorescence wavelength of 605 nm.
• the rate of fluid delivery of the culture medium dilution containing fluorescent beads and the culture medium not containing fluorescent beads was set to 500 [iL/min, the internal pressure of the upper microchannel was set to approximately 8.7 kPa, and the internal pressure of the lower microchannel was set to approximately 1.3 kPa so as to establish parameters close to the blood flow and blood pressure conditions inside actual blood vessels.
• a fluorescent bead permeability test was performed using the same method as the fluorescent bead permeability test for the cell-layer-affixed blood vessel models described above. Counts of the number of fluorescent beads inside the lower microchannel, namely, inside the culture medium, after a certain amount of time had elapsed since starting fluid delivery gave a number of fluorescent beads of 1.7> ⁇ 10 5 beads/ml in example 2, and a number of fluorescent beads of 6.7 ⁇ 10 3 beads/ml in comparative example 2.
• a cell-layer-affixed blood vessel model was produced with a layer of rat vascular endothelial cells formed on the upper face of the porous membrane and a layer of rat smooth muscle cells formed on the lower face of the porous membrane.
• a cell-layer-affixed blood vessel model was produced with a layer of rat vascular endothelial cells formed on the upper face of the porous membrane and a layer of rat smooth muscle cells formed on the lower face of the porous membrane.
• the cells employed in example 3 and comparative example 3 were the same as that employed in example 2 and comparative example 2.
• the lower microchannel was initially seeded with 100 ⁇ _, of a cell suspension of rat smooth muscle cells having a cell concentration of 3 ⁇ 10 6 cells/ml.
• the upper microchannel was seeded with 100 ⁇ ⁇ of a cell suspension of rat vascular endothelial cells having a cell concentration of 1 ⁇ 10 6 cells/ml and cultivated for six hours.
• a respective culture medium (rat EC medium/SMC medium) was run through each channel with a rate of fluid delivery of 0.7 ⁇ / ⁇ using a pump. After two days of cultivation, the
• vascular endothelial cell layer of the porous membrane of the cell-lay er-affixed blood vessel models produced in example 3 and comparative example 3 was exposed to a drug by running fenoldopam, this being the drug, through the upper microchannel at a concentration of 500 ng/ml and at flow rate of 0.7 ⁇ / ⁇ for one day.
• a FITC-Dextran permeability test was performed using the same method as the FITC-Dextran permeability test for the cell-layer-affixed blood vessel models described above. These results are illustrated in Fig. 9A and Fig. 9B.
• fluorescence was observed over a wide area, including in the lower microchannel.
• the fluorescence observed in the lower microchannel was minimal.
• porous membrane of the present exemplary embodiment enables evaluation of drug toxicity in a blood vessel model with high sensitivity.
• a blood vessel model was produced by providing 12 mm ⁇ 0.2 mm openings at straight portions of the upper and lower channels of the blood vessel model of example 1.
• the openings were formed by inserting a polypropylene reinforcing member, formed with a 0.2 mm wide slit, between the lower channel member and the porous membrane.
• the reinforcing member had a thickness of 100 ⁇ .
• Fig. lOA and Fig. 10B illustrates a micrograph of the porous membrane of example 4.
• a cell-lay er-affixed blood vessel model was produced by spraying collagen (5005-100ML, produced by Advanced BioMatrix) on the porous membrane of the blood vessel model of example 1 for 15 minutes at 60°C, and then allowing the collagen to dry so as to form a thick coating, and then forming a layer of rat vascular endothelial cells on the upper face of the porous membrane and forming a layer of rat smooth muscle cells on the lower face of the porous membrane.
• Fig. 11 illustrates a micrograph of the porous membrane of example 5.
• a blood vessel model with a single cell layer affixed thereto was produced by taking the blood vessel model of example 4 and forming a layer of rat vascular endothelial cells on the upper face of the porous membrane.
• a culture medium dilution containing fluorescent beads at a concentration of 1.81 x 10 6 beads/ml for a tracer was run through the upper microchannel of the
• the fluorescent beads had a diameter of 4 ⁇ and were labeled with red fluorescent dye with an excitation wavelength of 580 nm and a fluorescence wavelength of 605 nm.
• the rate of fluid delivery of the culture medium dilution containing fluorescent beads and the culture medium not containing fluorescent beads was set to 500 ⁇ ,/ ⁇ , the internal pressure of the upper microchannel was set to approximately 8.7 kPa, and the internal pressure of the lower microchannel was set to approximately 1.3 kPa so as to establish parameters close to the blood flow and blood pressure conditions inside actual blood vessels.
• a cell-lay er-affixed blood vessel model was produced by taking the blood vessel model of example 1 and forming a layer of human vascular endothelial cells derived from induced pluripotent stem cells on the upper face of the porous membrane and forming human mesenchymal stem cells on the lower face of the porous membrane.
• a porous membrane formed of polycarbonate similar to the porous membrane 28 of the above exemplary embodiment, was employed in which the openings of plural through-holes were arranged in a honeycomb pattern and the through-holes were in communication through communication holes.
• the average opening diameter of openings in the porous membrane of example 8 was 3 ⁇
• the opening coverage ratio of the openings was 52%
• the membrane thickness of the porous membrane was 1.2 ⁇
• the variation coefficient of the opening diameters of the openings was 5.0%
• the porosity of the porous membrane was 80%.
• a cell-lay er-affixed blood vessel model was produced by taking the blood vessel model of example 8 and forming rat vascular endothelial cells on the upper face of the porous membrane and forming rat smooth muscle cells the lower face of the porous membrane.

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