CN111263697A - Blood vessel model - Google Patents

Blood vessel model Download PDF

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Publication number
CN111263697A
CN111263697A CN201880037657.1A CN201880037657A CN111263697A CN 111263697 A CN111263697 A CN 111263697A CN 201880037657 A CN201880037657 A CN 201880037657A CN 111263697 A CN111263697 A CN 111263697A
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Prior art keywords
porous membrane
holes
microchannel
cell layer
porous
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CN201880037657.1A
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CN111263697B (en
Inventor
伊藤晃寿
柿沼千早
引本大地
美马伸治
末广贵史
C·M·内维尔
C·A·森德巴克
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Fujifilm Corp
General Hospital Corp
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Fujifilm Corp
General Hospital Corp
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Priority claimed from US15/618,151 external-priority patent/US20180356399A1/en
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
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    • G01N33/5064—Endothelial cells
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    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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Abstract

The present invention provides a blood vessel model, comprising: a pair of channel members facing each other and each including an opposing surface on which a respective channel is formed; and a porous membrane including a plurality of through-holes penetrating in a thickness direction, disposed between the facing surfaces of the pair of channel members, and partitioning the microchannel, wherein the porous membrane is provided with an intravascular endothelial cell layer so as to cover a surface facing one of the microchannels, an average opening diameter of the through-holes is 1 to 20 μm, and an opening coverage of the through-holes is 30 to 70%.

Description

Blood vessel model
Technical Field
The invention relates to a blood vessel model.
Background
In recent years, attempts have been made to simulate internal organs such as blood vessels, intestines, liver, and lungs by using a device including what is called a microchannel having a width on the order of micrometers. U.S. patent application publication (US) No. 2011/0053207, japanese patent application publication (JP-B) No. 5415538, and JP-B No. 5815643, for example, each disclose an internal organ model including a porous membrane having a cell layer on the surface thereof and at least two microchannels separated by the porous membrane.
Various tests and tests can be carried out using, for example, visceral organ models disclosed in US 2011/0053207, JP-B5415538 and JP-B5815643. For example, a so-called leak test can be performed by allowing blood containing a drug to flow through one microchannel, and then measuring the number or amount of red blood cells, biomarkers, and the like that move from one microchannel to another microchannel through a porous membrane. By this leakage test, the degree of drug-induced damage of the cell layer provided on the surface of the porous membrane can be evaluated, and a drug toxicology test can be performed.
However, the pores in the porous membrane used in the conventional organ model are produced by a known track etching process in which, for example, heavy ions are irradiated to the material constituting the porous membrane. Therefore, the opening coverage of the pores in the membrane is, for example, as low as 2% to 20%, and since the membrane is also thick, passage of red blood cells and the like is hindered by the porous membrane. That is, in the conventional organ model, the degree of drug damage of the cell layer provided on the surface of the porous membrane may not be accurately evaluated.
Disclosure of Invention
The present invention provides a blood vessel model that inhibits the migration of red blood cells and the like from being hindered by a porous membrane during a leak test.
A blood vessel model according to a first aspect of the present invention includes: a pair of channel members facing each other and each including an opposing surface on which a respective channel is formed; and a porous membrane including a plurality of through-holes penetrating in a thickness direction, disposed between the facing surfaces of the pair of channel members, and partitioning the microchannel, wherein the porous membrane has an intravascular endothelial cell layer to cover a surface facing one of the microchannels, an average opening diameter of the through-holes is 1 to 20 μm, and an opening coverage of the through-holes is 30 to 70%.
In the above structure, the average opening diameter of the through-holes in the porous membrane that partitions the microchannels is 1 to 20 μm, and the opening coverage of the through-holes is 30 to 70%. Therefore, when red blood cells or the like move from one microchannel to another microchannel while flowing through the through-holes in the porous membrane during the leak test, it is possible to suppress the movement of red blood cells or the like from being hindered by the porous membrane.
In a second aspect of the present invention, in the first aspect, the film thickness of the porous film may be equal to or less than half of the average opening diameter of the through-holes.
In the second aspect, since the thickness of the porous membrane is equal to or less than half of the average opening diameter of the through-holes, red blood cells and the like can more easily pass through the through-holes in the porous membrane, as compared with a case where the thickness of the porous membrane is larger than half of the average opening diameter of the openings of the through-holes. Therefore, the second approach may further improve the accuracy of the leak test.
In a third aspect of the present invention, in the first or second aspect, the communication holes that communicate the through-holes with each other may be formed inside the porous membrane; the through holes are arranged in a honeycomb shape; the coefficient of variation of the opening diameter of the through-hole may be 10% or less; and the porosity of the porous film may be 50% or more.
In the third mode described above, the through-holes are arranged in a honeycomb shape and communicate with each other through the communication holes. The coefficient of variation of the opening diameter of the opening of the through-hole is 10% or less, and the porosity of the porous membrane is 50% or more. In this way, in the third aspect, red blood cells and the like are more uniformly passed through. Therefore, the third mode can further improve the accuracy of the leak test.
In a fourth aspect of the present invention, in the first to third aspects, the cells of the cell layer may be selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes and fibroblasts, and the cell layer may be disposed on the other surface of the porous membrane opposite to the other microchannel.
In the fourth aspect, since the cell layer of the cell selected from the group consisting of smooth muscle cell, mesenchymal stem cell, pericyte and fibroblast is formed on the other surface of the porous membrane on the side opposite to the surface on which the vascular endothelial cell layer is formed, a blood vessel model closer to the actual blood vessel can be realized.
In a fifth aspect of the present invention, in the first to fourth aspects, the porous film may have a tensile elongation at break of 50% or more; and the stress required for 10% stretching of the porous film may be 1000gf/mm2The following.
In the fifth aspect, the porous film has a tensile elongation at break of 50% or more and hasHas a density of 1000gf/mm2The flexible material of the stress required for the following 10% stretch is formed, and thus a blood vessel model closer to an actual blood vessel can be realized.
In a sixth aspect of the present invention, in the first to fifth aspects, the through-hole may have a planar shape in a plan view, and may include a major axis and a minor axis.
In the sixth aspect, since the through-hole has a planar shape such as an elliptical shape in a plan view, red blood cells and the like can more easily pass through the through-hole. Therefore, the sixth mode can further improve the accuracy of the leak test.
In a seventh aspect of the present invention, in the first to sixth aspects, the porous membrane may include a porous region in which through-holes are formed and a non-porous region in which through-holes are not formed.
In the seventh aspect, for example, since the portions of the porous membrane disposed in the vicinity of the inlet and the vicinity of the outlet of the microchannel are configured as non-porous regions in which the through-holes are not formed, the flow of red blood cells and the like in the microchannel can be adjusted. Therefore, the seventh mode can further improve the accuracy of the leak test.
According to the above-described mode, the present invention can suppress the movement of red blood cells and the like from being restricted by the porous membrane during the leak test.
Drawings
Exemplary embodiments of the present invention will be described in detail with reference to the following drawings.
Fig. 1 is a perspective view showing an overall structure of a blood vessel model according to an exemplary embodiment.
Fig. 2 is an exploded perspective view showing the entire structure of a blood vessel model according to an exemplary embodiment.
Fig. 3 is an enlarged cross-sectional view showing a porous membrane of a blood vessel model according to an exemplary embodiment.
Fig. 4 is a plan view showing a porous membrane of a blood vessel model according to an exemplary embodiment.
Fig. 5 is a plan view of a porous membrane of a blood vessel model showing a modification.
Fig. 6 is a plan view of a porous membrane of a blood vessel model showing a modification.
Fig. 7A is a photomicrograph of the porous membrane of example 1.
Fig. 7B is a photomicrograph of the porous membrane of comparative example 1.
Fig. 8A is the image fluorescence results in the micro-channel of the cell layer adhesion blood vessel model of example 3.
Fig. 8B is the image fluorescence result in the micro channel of the cell layer adhesion blood vessel model of comparative example 3.
Fig. 9A is the result of FITC-dextran permeability test in the cell layer adhesion blood vessel model of example 3.
Fig. 9B is the result of FITC-dextran permeability test in the cell layer adhesion blood vessel model of comparative example 3.
Fig. 10A is a photomicrograph of the porous membrane of example 4.
Fig. 10B is a partially enlarged view of fig. 10A.
Fig. 11 is a photomicrograph of the porous membrane of example 5.
Detailed Description
Hereinafter, examples and modifications of exemplary embodiments of the present invention will be described with reference to fig. 1 to 6. The following exemplary embodiments are merely examples of the present invention, and do not limit the scope of the present invention. For convenience of explanation, the dimensions of the various structures in the drawings may be changed as appropriate. Thus, the scale of the drawings may be different from that in actual operation.
As shown in fig. 1 and 2, the blood vessel model 10 of the exemplary embodiment includes an upper passage member 12 and a lower passage member 14 which are stacked on each other. The upper channel member 12 and the lower channel member 14 are made of an elastic material such as Polydimethylsiloxane (PDMS), and have a substantially rectangular plate shape.
Examples of the material constituting the upper-layer channel member 12 and the lower-layer channel member 14 other than PDMS include cycloolefin polymer (COP), epoxy resin, polyurethane resin, styrene-based thermoplastic elastomer, olefin-based thermoplastic elastomer, acrylic-based thermoplastic elastomer, polyvinyl alcohol, and the like
The recess 18 defining the upper microchannel 16 is formed in the lower surface of the upper channel member 12, i.e., in the opposed surface 12A opposed to the lower channel member 14. The recess 18 includes an inlet 18A, an outlet 18B, and a passage portion 18C that communicates the inlet 18A with the outlet 18B.
The through holes 20A and 20B are formed in the upper-layer passage member 12 so as to penetrate through the upper-layer passage member 12 in the thickness direction, and have lower ends communicating with the inlet 18A and the outlet 18B, respectively. The upper ends of the through holes 20A and 20B open on the upper surface of the upper-layer passage member 12. A liquid supply tube (not shown) is connected to the upper ends of the through holes 20A and 20B.
Similarly, the recess 24 defining the lower microchannel 22 is formed in the upper surface of the lower channel member 14, i.e., in the opposite surface 14A opposite to the upper channel member 12. The recess 24 includes an inlet 24A, an outlet 24B, and a passage portion 24C that communicates the inlet 24A with the outlet 24B.
The inlet 24A and the outlet 24B of the lower passage member 14 and the inlet 18A and the outlet 18B of the upper passage member 12 are provided at positions not overlapping in a plan view. In contrast, the channel portion 24C of the lower-stage channel member 14 and the channel portion 18C of the upper-stage channel member 12 are provided at positions overlapping in a plan view.
Through holes 26A and 26B are also formed in the upper-stage passage member 12, penetrate the upper-stage passage member 12 in the thickness direction, and have lower ends communicating with the inlet 24A and the outlet 24B, respectively. The upper ends of the through holes 26A and 26B open on the upper surface of the upper-layer passage member 12. Liquid supply tubes (not shown) are connected to the upper ends of the through holes 26A and 26B.
The 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 bonded to the porous membrane 28 with being sandwiched therebetween. In addition to the bonding with an adhesive, various methods such as welding, suction (self-adhesion), or screwing may be used as a method for joining the upper tunnel member 12 and the lower tunnel member 14.
The porous film 28 is, for example, a hydrophobic polymer dissolved in a hydrophobic organic solvent. The hydrophobic organic solvent is a liquid having a solubility in water at 25 ℃ of 10(g/100g of water) or less.
Examples of the hydrophobic polymer include polymers such as polybutadiene, polystyrene, polycarbonate, polyester (e.g., polylactic acid, polycaprolactone, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polylactic acid-polycaprolactone copolymer, polyethylene terephthalate, polyethylene naphthalate, polyethylene glycol succinate, polybutylene succinate, and poly-3-hydroxybutyrate), polyacrylate, polymethacrylate, polyacrylamide (polyacrylamine), polymethacrylamide, polyvinyl chloride, polyvinylidene fluoride, polyhexafluoropropylene, polyvinyl ether, polyvinylcarbazole, polyvinyl acetate, polytetrafluoroethylene, polylactone, polyamide, polyimide, polyurethane, polyurea, polycyclic aromatic hydrocarbons (polyaromatics), polysulfone, polyethersulfone, polysiloxane derivative, and cellulose acylate (e.g., triethylcellulose, cellulose propionate, and cellulose acetate butyrate). From the viewpoint of producing a honeycomb film by using the production method disclosed in japanese patent No. 4,734,157, for example, a polymer dissolved in a hydrophobic organic solvent is preferable.
These polymers may have the form of a homopolymer, a copolymer, a polymer blend or a polymer alloy as required, for example, from the viewpoints of solubility in a solvent, optical characteristics, electrical characteristics, film strength and elasticity. These polymers may be used alone or in combination of two or more. The material of the porous membrane 28 is not limited to the hydrophobic polymer, and may be selected from the viewpoint of cell adhesion and the like.
The upper surface 28A and the lower surface 28B of the porous membrane 28 (hereinafter, the upper surface 28A and the lower surface 28B may be collectively referred to as "main surfaces") are set to a size that substantially covers the channel portions 18C and 24C of the upper layer microchannel 16 and the lower layer microchannel 22, thereby separating the upper layer microchannel 16 from the lower layer microchannel 22.
Specifically, the upper surface 28A of the porous membrane 28 (i.e., the major surface opposite the upper channel member 12) defines the upper microchannel 16 together with the recessed portion 18 of the upper channel member 12. The lower surface 28B of the porous membrane 28 (i.e., the major surface opposite the lower channel member 14) defines the lower microchannel 22 with the recesses 24 of the lower channel member 14.
As shown in fig. 3, for example, an intravascular endothelial cell layer 36 is disposed on the upper surface 28A of the porous membrane 28 to completely cover the upper surface 28A. Thereby, an environment very close to the inside of the blood vessel is formed inside the upper layer microchannel 16. Examples of vascular endothelial cells include: vascular endothelial cells derived from umbilical vein, umbilical artery, aorta, coronary artery, pulmonary microvasculature, or dermal microvasculature; and vascular endothelial cells differentiated from pluripotent stem cells.
The cell layer 38 is made of, for example, cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts, and is provided on the lower surface 28B of the porous membrane 28 so as to completely cover the lower surface 28B. Thus, the underlying microchannel 22 forms an environment very close to the exterior of the blood vessel. Mesenchymal Stem Cells (MSCs) are adult stem cells capable of dividing into myocytes, adipocytes, chondrocytes, and the like.
A cell layer 38 of cells selected from the group consisting of cellular smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts may be disposed on the upper surface 28A of the porous membrane 28, while a vascular endothelial cell layer 36 may be disposed on the lower surface 28B of the porous membrane 28. The vascular endothelial cell layer 36 may be provided on one main surface of the porous membrane 28. The cell layer 38 may not be provided on the other major surface of the porous membrane 28.
From the viewpoint of cell adhesion, at least one cell-seeded region of the upper surface 28A and the lower surface 28B of the porous membrane 28 is preferably coated with at least one selected from the group consisting of fibronectin, collagen (e.g., type I collagen, type IV collagen, or type V collagen), laminin, vitronectin, gelatin, perlecan, nidogen, proteoglycan, osteopontin, tenascin, rennin, basement membrane matrix, and polylysine. The porous film 28 and the through-holes 30 described later are preferably coated with at least one of these.
In order to provide the vascular endothelial cell layer 36 and the cell layer 38 on the main surface of the porous membrane 28, for example, the following methods can be used: the cell suspension is poured onto the upper microchannel 16 and the lower microchannel 22 to seed the cells on the major surface of the porous membrane 28. Further, the following method may be adopted: after cells are seeded and cultured on the main surface of the porous membrane 28 in a separate culture apparatus, the porous membrane 28 having the vascular endothelial cell layer 36 and the cell layer 38 formed thereon is attached to the blood vessel model 10.
As shown in fig. 3 and 4, a plurality of through-holes 30 penetrating the porous membrane 28 in the thickness direction are formed in the porous membrane 28. The openings 30A of the through-holes 30 are provided on the upper surface 28A and the lower surface 28B of the porous membrane 28, respectively. As shown in fig. 4, the opening 30A is circular in a plan view. The openings 30A are provided apart from each other. Flats 32 extend between adjacent openings 30A. The opening 30A is not limited to a circular shape, and may be formed in a polygonal shape.
The plurality of openings 30A are regularly arranged, for example, in a honeycomb shape as shown in fig. 4 in the present exemplary embodiment. The honeycomb array is an array in which the centers of the openings 30A are arranged at points at which the vertex positions and the diagonal lines intersect in parallel hexagons (preferably regular hexagons) or similar shapes. Here, "center of opening" means the center of the opening 30A in a plan view.
The arrangement of the openings 30A is not limited to the honeycomb shape. The openings 30A may be formed in a lattice shape or a face-centered lattice shape. The lattice-like arrangement is an arrangement in which the centers of the openings are arranged at the vertex positions in units of parallelograms (including squares, rectangles, and rhombuses, but preferably squares) or shapes similar thereto. The face-centered lattice array is an array in which the centers of the openings are arranged at points at which the vertex positions and the diagonals intersect each other in a parallelogram (including square, rectangle, and rhombus, but preferably square) or a shape similar thereto.
The arrangement of the openings 30A may be arbitrary. However, from the viewpoint of achieving a uniform density of the openings 30A on the upper surface 28A and the lower surface 28B of the porous film 28, it is preferable that the plurality of openings 30A are regularly arranged. The regular array is an array in which the coefficient of variation of the surface area of the array of the parallel hexagonal or parallelogram units is, for example, 10% or less. Some openings 30A may be missing or openings 30A may not be aligned. However, the openings 30A are preferably arranged continuously in all directions without interruption. The "coefficient of variation" is a value obtained by dividing the standard deviation of a given population by its average value, and is an index indicating the degree of dispersion in the population in%.
As shown in fig. 3, each of the through-holes 30 in the porous membrane 28 has a spherical segment shape in which the upper end and the lower end of a sphere are truncated. The through-holes 30 adjacent to each other communicate with each other through the communication holes 34 in the porous membrane 28.
Preferably, each through-hole 30 communicates with all adjacent through-holes 30. In the case where the openings 30A of the plurality of through-holes 30 are arranged in a honeycomb shape as in the present exemplary embodiment, each through-hole 30 communicates with six adjacent through-holes 30 through six communication holes 34. The through-holes 30 may have a cylindrical shape, a polygonal cylindrical shape, or the like, and the communication holes 34 may be tubular empty holes that connect adjacent through-holes 30.
The opening diameter D of each opening 30A of the through-hole 30 is, for example, a size through which red blood cells in blood can pass. Specifically, the average opening diameter is preferably 1 to 20 μm, and more preferably 3 to 10 μm. The average opening diameter is set to 1 μm or more, the through-holes 30 can have a size allowing red blood cells to pass therethrough, and the average opening diameter is set to 20 μm or less, so that the vascular endothelial cell layer 36 and the cell layer 38 can be held on the main surface of the porous membrane 28.
Where "opening diameter D" is the major axis of the opening 30A, and "average opening diameter" is the calculated average of the opening diameters D measured for an optional 10 or more openings 30A. "major axis" means the longest distance between arbitrarily selected two points on the outer contour of the opening. However, in the case where a direction is specified, "major axis" represents the longest distance between two points arbitrarily selected in the direction.
The opening coverage of the opening 30A of the through-hole 30 is preferably 30% to 70%, and more preferably 40% to 60%. When the opening coverage is 30% or more, the inhibition of the movement of the red blood cells by the porous membrane 28 can be suppressed, and when the opening coverage is 70% or less, the strength required for the porous membrane 28 can be achieved.
Where "opening coverage" represents the ratio of S2: S1 in%, where S1 represents the unit surface area of the porous membrane 28 assuming that the major surface of the porous membrane 28 is smooth (i.e., assuming that no openings 30A are present on the porous membrane 28), and S2 represents the total surface area of the openings 30A per unit surface area, where S1 and S2 use the same units of measure.
The film thickness T of the porous film 28 is preferably equal to or less than half the average opening diameter of the openings 30A of the through-holes 30. Specifically, the thickness T is preferably 0.5 to 10 μm, more preferably 1 to 10 μm. When the thickness T of the porous membrane 28 is set to a thickness of half or less of the average opening diameter of the through-holes 30, the movement of red blood cells can be suppressed from being hindered by the porous membrane 28.
Further, since the porous membrane 28 is a scaffold to which cells adhere and grow, as the opening coverage on the porous membrane 28 is higher and the membrane thickness of the porous membrane 28 is thinner, intercellular interaction between cells on one surface of the porous membrane 28 and cells on the other surface of the porous membrane 28, that is, at least one of information transmission by humoral factors or intercellular contact becomes more active. During the cell culture, the more active the intercellular interaction for providing the vascular endothelial cell layer 36 and the cell layer 38 on the main surface of the porous membrane 28 is, the more preferable the vascular model that functions close to the in vivo tissue can be made.
The coefficient of variation of the opening diameter D of the opening 30A is preferably 10% or less, and the smaller the coefficient of variation, the more preferable. The smaller the coefficient of variation of the opening diameter D, the more uniformly red blood cells and the like can pass through the plurality of through-holes 30 in the porous membrane 28.
The porosity of the porous membrane 28 is preferably 50% or more. When the porosity is 50% or more, the movement of red blood cells can be inhibited by the porous membrane 28. If the porosity is too large, the strength of the porous membrane 28 becomes insufficient for the strength required therefor, and therefore the porosity is preferably 95% or less.
Here, "porosity" means a ratio of V2: V1 in%, where V1 means a unit volume of the porous membrane 28 assuming that the main surface of the porous membrane 28 is smooth (i.e., assuming that the opening 30A does not exist in the porous membrane 28), and V2 means a total volume of the through-holes 30 and the communication holes 34 provided per unit volume, and V1 and V2 use the same measurement unit.
The tensile elongation at break of the porous film 28 is preferably 50% or more, more preferably 100%, and still more preferably 200% or more. The stress required for 10% stretch of the porous film 28 is preferably 1000gf/mm2The following. As the tensile elongation at break is increased and the stress required for 10% stretch is decreased, the material becomes more flexible. The porous membrane 28 can be bent, stretched and compressed to bring the vascular model 10 closer to the actual blood vessel.
Among them, the "tensile elongation at break" can be determined by the following method in accordance with JIS K6251: 2010 to measure the tensile elongation at break of the porous film 28. "stress required for 10% stretching" can be measured by the method according to JIS K6251: 2010 to measure the stress applied to the porous film 28 when the porous film 28 is stretched by 10%.
Examples of the method for producing the porous film 28 in which the through-holes 30 are formed include a nano-printing step, a condensation step, an etching step, a sandblasting step, and a compression molding step. The nano-printing process is as follows: the through-hole 30 is produced by pouring a material for constituting the porous membrane 28 into a mold having a convex portion and a concave portion or pressing the mold against the material constituting the porous membrane 28. The condensation process is as follows: the through-holes 30 are formed by using water droplets as a mold by inducing condensation on the surface of the material constituting the porous film 28.
Compared to other methods, the condensation step can make the film thickness of the porous film 28 thinner, and can improve the porosity and the opening coverage of the openings 30A, and can also provide the communication holes 34 in the porous film 28. Accordingly, in the present exemplary embodiment, the porous membrane 28 is produced by a condensation process. The condensation step is 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.
Next, as an example, a case where the vascular model 10 of the present exemplary embodiment is used to perform toxicological evaluation of a drug will be described. When a toxicology test for a drug is performed, first, the upper channel member 12 and the lower channel member 14 are bonded to the porous membrane 28 in a state of being sandwiched therebetween to produce the blood vessel model 10 including the upper microchannel 16 and the lower microchannel 22 as shown in fig. 2. The vascular endothelial cell layer 36 and the cell layer 38 are provided on the main surface of the porous membrane 28.
Thereafter, a blood diluent containing a drug is pumped into the upper microchannel 16 through the tube (not shown) and the through-hole 20A, passes through the inside of the upper microchannel 16, and is pumped out of the blood vessel model 10 through the through-hole 20B and the tube (not shown).
At the same time, the culture solution or the physiological saline solution is pumped into the lower microchannel 22 through the tube (not shown) and the through hole 26A, passes through the inside of the lower microchannel 22, and flows out of the blood vessel model 10 through the through hole 26B and the tube (not shown). The pressure in the upper microchannel 16 through which the blood diluent flows is higher than the pressure in the lower microchannel 22 through which the culture solution or physiological saline solution flows.
At the beginning of the toxicology test, as shown in fig. 3, the entire upper surface 28A and the entire lower surface 28B of the porous membrane 28 are covered by the vascular endothelial cell layer 36 and the cell layer 38, respectively. Thus, red blood cells in the blood cannot pass through the porous membrane 28 and do not leak to the underlying microchannel 22.
However, after a certain time has elapsed since the beginning of toxicology testing, the vascular endothelial cell layer 36 may be damaged by drug toxicity. In addition to the vascular endothelial cell layer 36, the cell layer 38 is also damaged by the drug. By measuring the flow of red blood cells into the underlying microchannel 22 through the porous membrane 28 due to these damaged portions, i.e., by performing a leak test, the degree of drug damage to the vascular endothelial cell layer 36 and the cell layer 38 can be evaluated.
When the vascular endothelial cell layer 36 is damaged by drug toxicity, the state of the cells constituting the cell layer 38 changes due to the interaction between the vascular endothelial cell layer 36 and the cell layer 38, and as a result, cracks may be generated in the cell layer 38. By measuring the red blood cells that pass through the gap and flow into the underlying microchannel 22, i.e., by performing a leak test, the degree of drug damage to the vascular endothelial cell layer 36 and the degree of reaction of the cell layer 38 can be evaluated.
The higher the opening coverage on the porous membrane 28 and the thinner the thickness of the porous membrane 28, the more active the intercellular interaction between the vascular endothelial cell layer 36 and the cell layer 38, and therefore the test can be performed with high sensitivity.
Moreover, in the toxicology test described above, the drug and the tracer-containing solution are circulated through the upper microchannel 16 instead of the blood diluent. Examples of the tracer include a fluorescent labeling chemical, a chemical containing a radioisotope, a dye compound, and the like, and more specifically, include at least one selected from the group consisting of dextran, evans blue, sodium fluorescein, and FITC microspheres. The fluorescent dye is preferably red with an excitation/fluorescence wavelength of 580nm/605 nm.
The degree of drug damage in the vascular endothelial cell layer 36 and the cell layer 38 can be evaluated by measuring the amount of the tracer flowing from the upper microchannel 16 into the lower microchannel 22 through the porous membrane 28, while measuring the fluorescence intensity, the radiation intensity, or the chromaticity according to the type of the tracer to quantify the tracer.
In the present exemplary embodiment, the upper microchannel 16 and the lower microchannel 22 are separated on the porous membrane 28, the average opening diameter of the openings 30A of the through-holes 30 is 1 μm to 20 μm, and the opening coverage of the openings 30A of the through-holes 30 is 30% to 70%. Therefore, when red blood cells flow through the upper microchannel 16 and move to the lower microchannel 22 through the through-holes 30 in the porous membrane 28 during the leak test, the movement of red blood cells can be inhibited from being hindered by the porous membrane 28.
In the present exemplary embodiment, the film thickness of the porous film 28 is equal to or less than half the average opening diameter of the openings 30A of the through-holes 30. Therefore, the red blood cells more easily pass through the through-holes 30 in the porous membrane 28 than in the case where the membrane thickness of the porous membrane 28 is larger than half the average opening diameter of the openings 30A of the through-holes 30. Thus, the present exemplary embodiment may further improve the accuracy of the leak test.
In the present exemplary embodiment, the openings 30A of the through-holes 30 are arranged in a honeycomb shape, and communicate with the through-holes 30 in the porous membrane 28 through the communication holes 34. The coefficient of variation of the opening diameter of the opening 30A of the through-hole 30 is 10% or less, and the porosity of the porous membrane 28 is 50% or more. Therefore, the red blood cells can more uniformly pass through the plurality of through-holes 30 in the porous membrane 28. Thus, the present exemplary embodiment may further improve the accuracy of the leak test.
In addition, the present exemplary embodiment is configured such that the vascular endothelial cell layer 36 is provided on the upper surface 28A of the porous membrane 28, and the cell layer 38 is provided on the lower surface 28B of the porous membrane 28. The cell layer 38 is composed of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts. The porous film 28 has a tensile elongation at break of 50% or more and a stress required for 10% stretching of 1000gf/mm2The following flexible materials. Thus, in the present exemplary embodiment, the blood vessel model 10 can be configured to be closer to the actual blood vessel.
Examples of exemplary embodiments of the present invention are described. However, the present invention is not limited to the above, and various modifications can be made without departing from the scope of the present invention.
For example, in the above exemplary embodiment, the opening 30A of the through-hole 30 in the porous membrane 28 is circular in a plan view, but as shown in fig. 5, the opening 50A of the through-hole 50 in the porous membrane 48 may be oval in a plan view. By configuring the opening 50A of the through-hole 50 to have an oval shape, for example, a disk-shaped red blood cell can easily pass through the opening 50A of the through-hole 50, while other cells in blood cannot easily pass through the opening 50A of the through-hole 50.
Examples of the method of forming the opening 50A of the through-hole 50 into an oval shape include a method of forming a circular opening 30A as shown in fig. 4 on the porous film 48, and then stretching the porous film 48 in one direction (the left-right direction in fig. 4). This method can form a plurality of elliptical openings 50A having a major axis direction in the same direction (left-right direction in fig. 5).
The oval-shaped opening 50A may be directly formed in the porous film 48 by compression molding or the like without stretching the porous film 48. In addition, as long as the shape of the opening 50A is a planar shape having a major axis and a minor axis in a plan view, the shape of the opening 50A may be, for example, a planar polygon obtained by stretching a regular polygon.
In the porous film 28 of the above exemplary embodiment, the openings 30A of the through-holes 30 are regularly arranged over the entire main surface of the porous film 28. However, as shown in fig. 6, the porous membrane 58 may have a porous region 62 in which the openings 60A of the through-holes 60 are formed and a non-porous region 64 (region marked with a two-dot chain line in fig. 6) in which the openings 60A of the through-holes 60 are not formed.
Specifically, the porous membrane 58 is configured as, for example, a non-porous region 64 in a portion disposed in the vicinity of the inlet 18A and the vicinity of the outlet 18B of the recess 18 constituting the upper microchannel 16 shown in fig. 1 and a portion disposed in the vicinity of the inlet 24A and the vicinity of the outlet 24B of the recess 24 constituting the lower microchannel 22 shown in fig. 1.
In general, the flow of fluid such as blood is easily obstructed at the inlets 18A and 24A and the outlets 18B and 24B. Therefore, by configuring the porous membrane 58 as the non-porous region 64 in the vicinity of the inlets 18A and 24A and in the vicinity of the outlets 18B and 24B, the flow of the liquid such as blood in the upper microchannel 16 and the lower microchannel 22 can be adjusted. Thus, the porous membrane 58 may further improve the accuracy of the leak test.
The blood vessel model of the present invention enables a leak test to be performed in a state in which the movement of a leaking substance such as red blood cells, which is associated with drug toxicity, to the outside of the blood vessel is inhibited by the porous membrane. Therefore, the blood vessel model of the present invention can be used as a blood vessel model capable of toxicological tests with high accuracy.
Hereinafter, examples of exemplary embodiments of the present invention will be described in detail. The exemplary embodiments of the invention should not be construed as being limited by the examples set forth below.
Fig. 7A shows a micrograph of the porous membrane of example 1. In example 1, a porous film formed of polycarbonate was used in the same manner as the porous film 28 of the exemplary embodiment, in which the openings of a plurality of through-holes were arranged in a honeycomb shape and the through-holes were communicated through the communication holes. In example 1, the average opening diameter of the openings of the porous membrane was 5 μm, the opening coverage of the openings was 55%, the membrane thickness of the porous membrane was 2.2 μm, the coefficient of variation of the opening diameter of the openings was 3.5%, and the porosity of the porous membrane wasThe film had a porosity of 75%, a tensile elongation at break of 250%, and a stress required for 10% stretching of 100gf/mm2。
The microstructure of the produced porous film was measured by using a contour scanning laser microscope (product name VK-X100, manufactured by Keyence, Japan). The observation was made with a magnification showing more than 50 apertures on one screen. From the observed micrographs, image analysis was performed on the openings present on one screen to measure each opening diameter D and find the average opening diameter DAV and the coefficient of variation σ D of the opening diameter D. The coefficient of variation (expressed in%) of the opening diameter can be obtained by calculating (σ D/DAV) × 100.
The average opening diameter and the opening coverage were obtained by subjecting the photomicrograph to binarization processing and image processing using 2D image analysis software WinROOF (Mitani Corp.). The film thickness of the porous film is an average value of the opening thicknesses measured at ten points by a profile scanning laser microscope.
The cross section of the porous membrane was observed by a scanning electron microscope (SEM, product name SU8030, manufactured by Hitachi, japan), and the diameter of a sphere equal to a through-hole was calculated as the porosity of the porous membrane. The porous membrane sample to be evaluated was cut with a microtome (product name FCS, manufactured by Reichert, austria) to prepare a sample for cross-section observation, the surface of the sample for cross-section observation was coated with an Os layer having a thickness of 6nm, and the sample was observed with SEM at an acceleration voltage of 2 kV. Tensile elongation at break and stress required for 10% stretching of the porous film were measured by FUDOH RHEO METERRT-2002 D.D. (manufactured by Rheotech Corp.).
Fig. 7B shows a micrograph of the porous membrane of comparative example 1. In comparative example 1, a porous film of a conventional technique formed of polycarbonate in which an opening was formed by a track etching process was used. In comparative example 1, the average opening diameter of the openings of the porous film was 5.7 μm, the opening coverage of the openings was 12.4%, the film thickness of the porous film was 10.6 μm, the coefficient of variation of the opening diameter of the openings was 35%, the porosity of the porous film was 15%, the tensile elongation at break was 150%, and the stress required for 10% stretching was 5800gf/mm2。
The porous film is prepared by attaching medical paper to both sides thereof. The medical paper on one surface of the porous membrane is removed with tweezers, and the surface from which the medical paper is removed is placed on the lower channel member facing downward. The porous membrane and the lower channel member were then joined by immersing the porous membrane in ethanol using a swab.
Next, the medical paper on the other surface of the porous membrane was removed with tweezers, and the upper channel member was laminated on the other surface of the porous membrane. The positions of the upper and lower channel members were aligned while confirming under a microscope, and the upper and lower channel members were joined. Thus, the blood vessel model of example 1 and the blood vessel model of comparative example 1 were prepared.
In example 1 and comparative example 1, in order to evaluate the permeability of the porous membrane to red blood cells, the porous membrane used did not have the vascular endothelial cell layer 36 or a cell layer of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes and fibroblasts, provided on the main surface thereof.
In example 2, a blood vessel model with a cell layer attached thereto was prepared by forming a rat vascular endothelial cell layer on the upper surface of the porous membrane and a cell layer composed of rat smooth muscle cells on the lower surface of the porous membrane using the blood vessel model of example 1.
In comparative example 2, a blood vessel model with a cell layer attached thereto was prepared by forming a rat vascular endothelial cell layer on the upper surface of the porous membrane and a cell layer composed of rat smooth muscle cells on the lower surface of the porous membrane using the blood vessel model of comparative example 1.
In example 2 and comparative example 2, rat arterial endothelial cells made by Angio-Proteomie were used as rat vascular endothelial cells, and rat aortic smooth muscle cells made by Lonza were used as rat smooth muscle cells. The lower microchannel was initially seeded with a cell concentration of 3X 106Cell suspension of rat smooth muscle cells at cell/ml 100. mu.L. One day after the culture, the upper microchannel was seeded with cells at a concentration of 3X 106Cell suspension of rat vascular endothelial cells at cell/ml 100. mu.L. After two days of culture, cell layer-adherent blood vessel models of example 2 and comparative example 2 were obtained.
The number of red blood cells is 3.7X 105The blood diluent of cell/ml was passed through the upper microchannel of the blood vessel model fabricated in example 1 and comparative example 1, and the physiological saline solution was passed through the lower microchannel thereof. Parameters approximating the blood flow and blood pressure conditions in the actual blood vessel were set by setting the liquid transport rate of the blood diluent and the physiological saline solution to 500 μ L/min, the internal pressure of the upper microchannel to about 8.7kPa, and the internal pressure of the lower microchannel to about 1.3 kPa.
Regarding the amount of red blood cells in the lower microchannel, i.e., in the physiological saline solution, the number of red blood cells in example 1 was 9.2X 10 after a certain time had elapsed from the start of liquid transfer4Cells/ml, and the number of erythrocytes in the blood vessel model of comparative example 1 was 2.2X 104Cells/ml.
This test confirmed that the porous membranes of example 1 and comparative example 1 both have permeability to red blood cells under conditions equivalent to blood pressure conditions. Furthermore, since red blood cells more easily pass through the porous membrane of example 1 than the porous membrane of comparative example 1, it was confirmed that the porous membrane of the present exemplary embodiment can suppress the inhibition of the migration of red blood cells.
So as to make the inclusion concentration be 1.81X 106A medium diluent of fluorescent microspheres as a tracer in a sphere/ml was passed through the upper microchannel of the cell layer-adhered vascular model fabricated in example 2 and comparative example 2, and a medium containing no fluorescent microspheres was passed through the lower microchannel. The fluorescent microspheres had a diameter of 4 μm and were labeled with a red fluorescent dye having an excitation wavelength of 580nm and a fluorescence wavelength of 605 nm. Parameters approximating the blood flow and blood pressure conditions in the actual blood vessel were set by setting the liquid transport rates of the medium dilution containing fluorescent microspheres and the medium containing no fluorescent microspheres to 500. mu.L/min, the internal pressure of the upper microchannel to about 8.7kPa, and the internal pressure of the lower microchannel to about 1.3 kPa.
Regarding the number of fluorescent microspheres in the lower microchannel, i.e., in the culture medium, the number of fluorescent microspheres in example 2 was 6.5X 10 after a certain time from the start of liquid transfer4Ball/ml, and number of fluorescent microspheres in comparative example 2Is 9.2X 103Ball/ml. So as to be 1.81 multiplied by 106The diluted saline solution containing fluorescent microspheres in beads/ml was passed through the microchannels of the blood vessel models prepared in example 1 and comparative example 1, and the saline solution was passed through the lower layer of the microchannels. The liquid transfer rate was set to 500. mu.L/min. As for the number of fluorescent microspheres in the lower microchannel, the number of fluorescent microspheres in example 1 was 1.7X 105Ball/ml, whereas the fluorescent microspheres in comparative example 1 were 4.3X 104Ball/ml. This test confirmed that the formation of cell layers on both sides of the porous membrane reduced the permeability of the porous membrane to the fluorescent microspheres and imparted barrier properties to the porous membrane.
The cell layers on both sides of the porous membrane of the cell layer-adhered vascular model prepared in example 2 and comparative example 2 were exposed to the drug by allowing the drug, cytochalasin, to flow through the upper-layer microchannel and the lower-layer microchannel at a concentration of 50 μ g/ml and a flow rate of 0.7 μ L/min for 1 day.
After drug exposure, the fluorescent microsphere permeability test was performed using the same method as the fluorescent microsphere permeability test for the cell layer-adhered vascular model described above. Regarding the number of fluorescent microspheres in the lower microchannel, i.e., in the culture medium, the number of fluorescent microspheres in example 2 was 1.7X 10 after a certain time from the start of liquid transfer5Spheres/ml, whereas the number of fluorescent microspheres in comparative example 2 was 6.7X 103Ball/ml.
This test confirmed that the fluorescent microspheres were able to pass through the porous membrane in the cell layer-adhered vascular models of example 2 and comparative example 2 after the cell layer was damaged by the drug. Further, since the fluorescent microspheres were able to pass through the porous film of example 2 more than the porous film of comparative example 2, it was confirmed that the porous film of the present exemplary embodiment can suppress the movement of the fluorescent microspheres from being hindered. Thus, it was confirmed that the porous membrane of the present exemplary embodiment can evaluate drug toxicity with high sensitivity in a blood vessel model.
In example 3, a cell layer-adhered blood vessel model having a rat vascular endothelial cell layer formed on the upper surface of the porous membrane and a rat smooth muscle cell layer formed on the lower surface of the porous membrane was prepared in the same manner as in example 2.
In comparative example 3, a cell layer-adhered blood vessel model having a rat vascular endothelial cell layer formed on the upper surface of the porous membrane and a rat smooth muscle cell layer formed on the lower surface of the porous membrane was prepared in the same manner as in example 2.
The cells used in example 3 and comparative example 3 were the same as those used in example 2 and comparative example 2. To form the cell layer, the lower microchannel was initially seeded with cells at a concentration of 3X 106Cell suspension of 100. mu.L of rat smooth muscle cells per ml. After one day of culture, the upper microchannel was seeded with cells at a concentration of 1X 106Cells/ml of rat vascular endothelial cells 100. mu.L of cell suspension and cultured for 6 hours. Then, each medium (rat EC medium/SMC medium) was flowed through each channel at a liquid delivery rate of 0.7 μ L/min using a pump. After two days of culture, cell layer-adherent blood vessel models of example 3 and comparative example 3 were obtained.
The lower microchannel of the cell layer-adhered vascular model prepared in example 3 and comparative example 3 was closed, and a medium diluent containing FITC-dextran (46945, manufactured by Sigma-Aldrich) as a tracer at a concentration of 12.5. mu.g/50. mu.l was passed through the upper microchannel. The liquid delivery rate of the medium dilution containing FITC-dextran was set at 7 μ L/min.
After allowing FITC-dextran to flow through the channel for 2 minutes, fluorescence of the microchannel was imaged using an inverted microscope (product name eclipse Ts2, manufactured by Nikon). As for the imaging parameters, 4-fold magnification, 1600-fold gain, 60ms exposure time were used. These results are shown in fig. 8A and 8B. In example 3 and comparative example 3, no fluorescence was observed in the lower channel. This indicates that FITC-dextran did not permeate from the upper channel to the lower channel. This test can confirm that the formation of cell layers on both sides of the porous membrane hinders FITC-dextran permeation and imparts a barrier function to the porous membrane.
The upper microchannel was flowed through by fenoldopam (fenoldopam) which was a drug at a concentration of 500ng/ml and a flow rate of 0.7 μ L/min over 1 day, thereby exposing the vascular endothelial cell layer of the porous membrane of the cell layer-adhered vascular model fabricated in example 3 and comparative example 3 to the drug.
After drug exposure, the FITC-dextran permeability test was performed using the same method as the FITC-dextran permeability test for the cell layer adhesion blood vessel model described above. These results are shown in fig. 9A and 9B. In the cell layer-adhered blood vessel model of example 3, fluorescence was observed in a wide range including the lower microchannel in addition to the upper microchannel. In the cell layer adhesion vessel model of comparative example 3, minimal fluorescence was observed in the lower microchannel. This test confirmed that after the cell layer was damaged by the drug, FITC-dextran could pass through the porous membrane in the cell layer adhesion blood vessel models of example 3 and comparative example 3. Further, the porous membrane of example 3 had higher permeability to FITC-dextran than the porous membrane of comparative example 3, and it could be confirmed that the porous membrane of the present exemplary embodiment did not inhibit migration of FITC-dextran. Thus, it was confirmed that the porous membrane of the present exemplary embodiment can evaluate drug toxicity with high sensitivity in a blood vessel model.
In example 4, a blood vessel model was prepared by providing openings of 12mm × 0.2mm in the 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 0.2mm wide slits between the lower channel member and the porous membrane. The reinforcing member has a thickness of 100 μm. Fig. 10A and 10B show micrographs of the porous membrane of example 4.
In example 5, collagen (5005-100ML, manufactured by Advanced biometrix) was sprayed at 60 ℃ for 15 minutes on the porous membrane of the blood vessel model of example 1, and then dried to form a thick coating, and then an intravascular endothelial cell layer was formed on the upper surface of the porous membrane and rat smooth muscle cells were formed on the lower surface of the porous membrane, thereby forming a cell layer-adhered blood vessel model. FIG. 11 shows a photomicrograph of the porous membrane of example 5.
In example 6, using the blood vessel model of example 4, a rat vascular endothelial cell layer was formed on the upper surface of the porous membrane to form a blood vessel model to which a single cell layer was adhered.
So as to make the inclusion concentration be 1.81X 106Fluorescent microspheres of ball/ml as indicationThe diluent medium of the tracer was circulated through the upper microchannel of the blood vessel model to which the single cell layer was adhered, which was prepared in example 6, and the medium containing no fluorescent microspheres was circulated through the lower microchannel of the blood vessel model. The fluorescent microspheres were 4 μm in diameter and were labeled with a red fluorescent dye having an excitation wavelength of 580nm and a fluorescence wavelength of 605 nm. Parameters approximating the blood flow and blood pressure conditions in the actual blood vessel were set by setting the liquid transport rates of the medium dilution containing fluorescent microspheres and the medium containing no fluorescent microspheres to 500. mu.L/min, the internal pressure of the upper microchannel to about 8.7kPa, and the internal pressure of the lower microchannel to about 1.3 kPa.
Regarding the number of fluorescent microspheres in the lower microchannel, i.e., in the culture medium, the number of fluorescent microspheres in example 6 was 2.67X 10 after a certain time from the start of liquid transfer4Ball/ml. So as to be 1.81 multiplied by 106A saline diluent containing fluorescent microspheres in a volume of beads/ml was passed through the upper channel of the vascular model of example 4, and saline was passed through the lower channel at a fluid delivery rate of 500. mu.L/min, thereby obtaining a volume of 7.23X 10 in example 45Number of fluorescent microspheres per ml. This test confirmed that the formation of a cell layer on one surface of the porous membrane reduced the permeability of the porous membrane to the fluorescent microspheres and imparted barrier properties to the porous membrane.
In example 7, using the blood vessel model of example 1, a cell layer-adhering blood vessel model was prepared by forming a human vascular endothelial cell layer derived from induced pluripotent stem cells on the upper surface of the porous membrane and forming human mesenchymal stem cells on the lower surface of the porous membrane.
In example 8, a porous film was used in which the openings of a plurality of through-holes were arranged in a honeycomb shape, and the through-holes were communicated through communication holes, and which was formed of polycarbonate, similarly to the porous film 28 of the exemplary embodiment. In example 8, the average opening diameter of the openings of the porous membrane was 3 μm, the opening coverage of the openings was 52%, the membrane thickness of the porous membrane was 1.2 μm, the coefficient of variation of the opening diameter of the openings was 5.0%, and the porosity of the porous membrane was 80%.
In example 9, using the blood vessel model of example 8, rat vascular endothelial cells were formed on the upper surface of the porous membrane, and rat smooth muscle cells were formed on the lower surface of the porous membrane, thereby preparing a cell layer-adhering blood vessel model.

Claims (8)

1. A vascular model, comprising:
a pair of channel members facing each other and each including an opposing face on which a respective microchannel is formed; and
a porous membrane including a plurality of through-holes penetrating in a thickness direction, disposed between the facing surfaces of the pair of channel members, and partitioning the microchannel,
and providing an intravascular endothelial cell layer on the porous membrane to cover a surface opposite to one of the microchannels, wherein the average opening diameter of the through-holes is 1 to 20 μm, and the opening coverage of the through-holes is 30 to 70%.
2. The vessel model of claim 1,
the film thickness of the porous film is not more than half of the average opening diameter of the through-holes.
3. The vessel model of claim 1, further comprising:
communication holes formed inside the porous membrane to communicate the through-holes with each other,
the through-holes are arranged in a honeycomb shape,
the coefficient of variation of the opening diameter of the through-hole is 10% or less,
the porosity of the porous film is 50% or more.
4. The blood vessel model of claim 1, further comprising a cell layer disposed on another surface of the porous membrane opposite to another microchannel, the cells of the cell layer being selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes and fibroblasts.
5. The vessel model of claim 1,
the porous film has a tensile elongation at break of 50% or more,
the stress required for 10% stretching of the porous film is 1000gf/mm2The following.
6. The vessel model of claim 1,
the through-hole has a planar shape in a plan view and includes a major axis and a minor axis.
7. The vessel model of claim 1,
the porous membrane includes a porous region in which through-holes are formed and a non-porous region in which through-holes are not formed.
8. A method of leak testing using a blood diluent containing a drug, the method comprising:
providing the blood vessel model of claim 1;
a step of allowing a blood diluent containing a drug to flow through a microchannel facing the surface of the porous membrane provided with the vascular endothelial cell layer; and
and counting the number of red blood cells leaked to the microchannel facing the other surface of the porous membrane.
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