JP6869379B2 - Blood vessel model - Google Patents

Description

The present disclosure relates to a vascular model.

Recently, there have been attempts to model organs such as blood vessels, internal organs, liver and lungs using devices that include what is called a microchannel, which is a channel with a width in the order of micrometers. For example, U.S. Patent Application No. 2011/0053207, Japanese Patent No. 5415538, and Japanese Patent No. 5815643 each have a porous membrane having a cell layer on its surface and at least two defined by the porous membrane. An organ model containing two microchannels is disclosed.

A variety of experiments and tests can be performed using organ models such as those disclosed in US Patent Application 2011/0053207, Japanese Patent 5415538, and Japanese Patent 5815643. For example, measuring the number or amount of red blood cells, biomarkers, etc. that have moved from one microchannel to another through a perforated membrane after flowing drug-containing blood through one of the microchannels. A test called an extension test can be performed at the site. This bleeding evaluation makes it possible to evaluate the level of drug-induced damage to the cell layer provided on the surface of the porous membrane, and a drug toxicity test can be performed.

However, the pores of the porous membrane used in the conventional organ model are generated by a process known as a track etching method, in which, for example, a substance constituting the porous membrane is irradiated with heavy ions. Therefore, the aperture ratio of the pores of the membrane is low, for example, 2% to 20%, and since the membrane is thick, the passage of red blood cells and the like is hindered by the porous membrane. That is, in the conventional organ model, the level of drug-induced damage to the cell layer provided on the surface of the porous membrane may not be accurately evaluated.

The present disclosure provides a vascular model capable of suppressing the movement of erythrocytes and the like from being blocked by a porous membrane during bleeding evaluation.

The blood vessel model according to the first aspect of the present disclosure includes a pair of flow path members facing each other including facing surfaces on which each micro flow path is formed; and a pair of through holes penetrating in the thickness direction. Containing a porous membrane that is disposed between the opposing surfaces of the flow path members and defines between the microchannels, where the porous membrane is provided with a vascular endothelial cell layer and faces one of the microchannels. The average opening diameter of the through hole is 1 μm to 20 μm, and the opening ratio of the through hole is 30% to 70%.

In the above configuration, the average opening diameter of the through-holes of the porous membrane defining between the microchannels is 1 μm to 20 μm, and the opening ratio of the through-holes is 30% to 70%. Therefore, during bleeding evaluation, when erythrocytes and the like move through the through holes of the porous membrane and move from one of the microchannels to the other one of the microchannels, the movement of erythrocytes and the like moves through the porous membrane. It can be suppressed from being hindered by.

In the second aspect of the present disclosure, in the first aspect, the film thickness of the porous membrane may be half or less of the average opening diameter of the through holes.

In the second aspect, since the film thickness of the porous membrane is less than half the average opening diameter of the through hole, the erythrocyte is larger than half the average opening diameter of the opening of the through hole. Etc. can more easily pass through the through holes of the porous membrane. Therefore, the second aspect can further improve the accuracy of the bleeding evaluation.

In the third aspect of the present disclosure, in the first or second aspect, the communication holes for communicating the through holes with each other may be formed inside the porous membrane; the through holes may be arranged in a honeycomb shape; The coefficient of variation of the opening diameter of the through hole may be 10% or less; the porosity of the porous membrane may be 50% or more.

In the third aspect, 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 through-hole opening is 10% or less, and the porosity of the porous membrane is 50% or more. Thereby, in the third aspect, red blood cells and the like can be passed more uniformly. Therefore, the third aspect can further improve the accuracy of the bleeding evaluation.

In the fourth aspect of the present disclosure, in the first to third aspects, the cell layer of the cell can be selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts, and one other. It may be provided on another surface of the porous film facing the microchannel.

In the fourth aspect, cells derived from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts are formed on the other surface of the porous membrane opposite to the surface on which the vascular endothelial cell layer is formed. Due to the formation of cell layers, vascular models that are more similar to real blood vessels can be achieved.

In the fifth aspect of the present disclosure, in the first to fourth aspects, the tensile elongation at break of the porous membrane is 50% or more; the stress required for 10% elongation of the porous membrane is 1000 gf / mm ² or less. You may.

In the fifth aspect, the porous membrane is formed from a flexible material having a tensile elongation at break of 50% or more and a stress required for elongation of 10% of 1000 gf / mm ² or less, and thus is an actual blood vessel. A more similar vascular model can be achieved.

In the sixth aspect of the present disclosure, in the first to fifth aspects, the through hole may have a flat 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 flat shape such as an ellipse in a plan view, red blood cells and the like can pass through the through hole more easily. Therefore, the sixth aspect can further improve the accuracy of the bleeding evaluation.

In the seventh aspect of the present disclosure, in the first to sixth aspects, the porous membrane can include a porous region in which a through hole is formed and a non-porous region in which a through hole is not formed.

In the seventh aspect, for example, a part of the porous membrane arranged near the inlet and the outlet of the microchannel is configured as a non-porous region in which no through hole is formed, so that the inside of the microchannel is formed. It can regulate the flow of red blood cells and the like. Therefore, the seventh aspect can further improve the accuracy of the bleeding evaluation.

According to the above aspect, the present disclosure can suppress the movement of red blood cells and the like during bleeding evaluation from being hindered by the porous membrane.

It is a perspective view which shows the whole structure of the blood vessel model of an exemplary embodiment. It is an exploded perspective view which shows the whole structure of the blood vessel model of an exemplary embodiment. FIG. 5 is an enlarged cross-sectional view showing a porous membrane of a blood vessel model of an exemplary embodiment. It is a top view which shows the porous membrane of the blood vessel model of an exemplary embodiment. It is a top view which shows the porous membrane of the blood vessel model of a modification. It is a top view which shows the porous membrane of the blood vessel model of a modification. It is a micrograph of the porous membrane of Example 1. It is a micrograph of the porous membrane of Comparative Example 1. It is the result of the image fluorescence in the microchannel of the cell layer attachment blood vessel model of Example 3. This is the result of image fluorescence in the microchannel of the cell layer-attached blood vessel model of Comparative Example 3. It is the result of the FITC-dextran permeability test in the cell layer attachment blood vessel model of Example 3. It is the result of the FITC-dextran permeability test in the cell layer attachment blood vessel model of Comparative Example 3. It is a micrograph of the porous membrane of Example 4. FIG. 10A is a partially enlarged view. It is a micrograph of the porous membrane of Example 5.

Examples and modifications of the exemplary embodiments of the present disclosure will be described with reference to FIGS. 1-6. The following exemplary embodiments are merely examples of the present disclosure and do not limit the scope of the present disclosure. Also, the dimensions of the various configurations in the drawings are modified as appropriate to facilitate the various configurations. Therefore, the scale of the drawing may differ from the actual scale.

As shown in FIGS. 1 and 2, the blood vessel model 10 of the exemplary embodiment includes an upper channel member 12 and a lower channel member 14 stacked on top of each other. The upper flow path member 12 and the lower flow path member 14 are made of an elastic substance such as polydimethylsiloxane (PDMS) and have a substantially rectangular plate shape.

In addition to PDMS, other examples of materials constituting the upper flow path member 12 and the lower flow path member 14 include cycloolefin polymer (COP), epoxy resin, urethane resin, styrene-based thermoplastic elastomer, and olefin-based thermoplastic. Includes elastomers, acrylic thermoplastic elastomers, polyvinyl alcohol, etc.

The recess 18 defining the upper microchannel 16 is formed on the lower surface of the upper channel member 12, that is, on the facing surface 12A facing the lower channel member 14. The recess 18 includes an inlet 18A, an outlet 18B, and a flow path portion 18C that communicates the inlet 18A and the outlet 18B.

The through holes 20A and 20B are formed in the upper flow path member 12, and have a lower end portion that penetrates the upper flow path member 12 in the thickness direction and communicates with the inlet 18A and the outlet 18B, respectively. The upper ends of the through holes 20A and 20B are open to the upper surface of the upper flow path member 12. A liquid supply pipe (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 on the upper surface of the lower channel member 14, that is, on the facing surface 14A facing the upper channel member 12. The recess 24 includes an inlet 24A, an outlet 24B, and a flow path portion 24C that communicates the inlet 24A and the outlet 24B.

The inlet 24A and outlet 24B of the lower flow path member 14 and the inlet 18A and outlet 18B of the upper flow path member 12 are provided at positions where they do not overlap in a plan view. On the other hand, the flow path portion 24C of the lower flow path member 14 and the flow path portion 18C of the upper flow path member 12 are provided at overlapping positions in a plan view.

Through holes 26A and 26B are also formed in the upper flow path member 12, and have a lower end portion that penetrates the upper flow path member 12 in the thickness direction and communicates with the inlet 24A and the outlet 24B, respectively. The upper ends of the through holes 26A and 26B are open to the upper surface of the upper flow path member 12. A liquid supply pipe (not shown) is connected to the upper ends of the through holes 26A and 26B.

The porous membrane 28 is provided between the facing surfaces 12A and 14A of the upper flow path member 12 and the lower flow path member 14. The upper flow path member 12 and the lower flow path member 14 are joined to the porous membrane 28 in a state of being sandwiched between them. Further, as a method of joining the upper flow path member 12 and the lower flow path member 14, in addition to the method of joining using an adhesive, various methods such as welding, adsorption (self-adsorption), or joining with a bolt can be used. Can be adopted.

The porous membrane 28 is, for example, a hydrophobic polymer dissolved in a hydrophobic organic solvent. The hydrophobic organic solvent is a liquid having a solubility of 10 or less (g / 100 g water) in water at 25 ° C.

Examples of hydrophobic polymers are polybutadiene, polystyrene, polycarbonate, polyester (eg, 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, polymethacrylicamide, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyhexafluoro. Propen, polyvinyl ether, polyvinyl carbazole, polyvinyl acetate, polytetrafluoroethylene, polylactone, polyamide, polyimide, polyurethane, polyurea, polycyclic aromatic compounds, polysulfone, polyethersulfone, polysiloxane derivatives, and cellulose acetate (eg, tri). Contains polymers such as acetyl cellulose, cellulose acetate propionate, and cellulose acetate butyrate). For example, from the viewpoint of producing a honeycomb film by using the production method disclosed in Japanese Patent No. 4734157, a polymer dissolved in a hydrophobic organic solvent is preferable.

For example, in terms of solubility in solvents, optical properties, electrical properties, film strength, and elasticity, these polymers can take the form of homopolymers, copolymers, polymer blends or polymer alloys as needed. 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 various materials can 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 surface”) are the flow path portions of the upper micro flow path 16 and the lower micro flow path 22. The size is determined so as to substantially cover 18C and 24C, and the upper microchannel 16 is defined from the lower microchannel 22.

Specifically, the upper surface 28A of the porous membrane 28, that is, the main surface facing the upper flow path member 12, defines the upper micro flow path 16 together with the recess 18 of the upper flow path member 12. The lower surface 28B of the porous membrane 28, that is, the main surface facing the lower flow path member 14, defines the lower micro flow path 22 together with the recess 24 of the lower flow path member 14.

As shown in FIG. 3, for example, the vascular endothelial cell layer 36 is provided on the upper surface 28A of the porous membrane 28 and completely covers the upper surface 28A. As a result, the inside of the upper microchannel 16 constitutes an environment very similar to the inside of the blood vessel. Examples of vascular endothelial cells include umbilical veins, umbilical arteries, aortas, coronary arteries, pulmonary arteries, pulmonary microvessels, or vascular endothelial cells derived from dermal microvessels; and vascular endothelial cells differentiated from pluripotent stem cells.

For example, a cell layer 38 composed of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts is provided on the lower surface 28B of the porous membrane 28 to completely complete the lower surface 28B. Cover with. As a result, the lower microchannel 22 constitutes an environment very similar to the outside of the blood vessel. Mesenchymal stem cells (MSCs) are somatic stem cells that can divide into muscle cells, adipocytes, chondrocytes, and the like.

The upper surface 28A of the porous membrane 28 may be provided with a cell layer 38 of cells selected from the group consisting of smooth muscle cells, mesenchymal stem cells, pericytes, and fibroblasts, and the porous membrane 28 may be provided with a cell layer 38. The lower surface 28B may be provided with a vascular endothelial cell layer 36. Further, it is sufficient that the vascular endothelial cell layer 36 is provided on at least one of the main surfaces of the porous membrane 28. The other main surface of the porous membrane 28 may be configured so that the cell layer 38 is not provided.

From the viewpoint of cell adhesion, the region where cells are seeded on at least one of the upper surface 28A and the lower surface 28B of the porous membrane 28 is fibronectin, collagen (for example, type I collagen, type IV collagen or type V collagen). , Laminin, Vitronectin, Gelatin, Perlecan, Nidozen, Proteoglycan, Osteopontin, Tenesin, Nephronectin, Basement Membrane Matrix, and Polylysine, preferably coated with at least one selected from the group. Further, it is preferable that the inside of the porous membrane 28 and the through hole 30 described later is coated with at least one of these.

In order to provide the vascular endothelial cell layer 36 and the cell layer 38 on each main surface of the porous membrane 28, for example, a cell suspension is poured into the upper microchannel 16 and the lower microchannel 22 and is placed on the main surface of the porous membrane 28. A method of seeding cells can be adopted. Further, there is also a method in which cells are seeded and cultured on the main surface of the porous membrane 28 in another culture apparatus, and then the porous membrane 28 on which the vascular endothelial cell layer 36 and the cell layer 38 are formed is attached to the blood vessel model 10. Can be adopted.

As shown in FIGS. 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. An opening 30A of the through hole 30 is provided on each of the upper surface 28A and the lower surface 28B of the porous membrane 28. As shown in FIG. 4, the opening 30A is circular in a plan view. The openings 30A are provided separately from each other. The flat portion 32 extends between adjacent openings 30A. The opening 30A is not limited to a circular shape, and may be formed of a polygonal shape.

The plurality of openings 30A are regularly arranged, and in this exemplary embodiment, they are arranged, for example, in a honeycomb shape, as shown in FIG. The honeycomb-shaped array is an array in which the center of the opening 30A is arranged at a point where the position of the apex and the diagonal line intersect with respect to a unit of a parallel hexagon (preferably a regular hexagon) or a shape close thereto. Here, the "center of the 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 opening 30A can be configured in a lattice pattern or a face-centered lattice pattern. A grid-like array is an array in which the center of the opening is arranged at the position of the apex with respect to a unit having a parallelogram (preferably a square, not to mention a square, a rectangle, and a rhombus) or a unit having a shape close to the parallelogram. The face-to-center grid arrangement is such that the center of the opening is a parallelogram (preferably a square, not to mention squares, rectangles, rhombuses) or at the point where the vertices and diagonals intersect with respect to a unit of similar shape. It is an array to be arranged.

The arrangement of openings 30A may be arbitrary. However, from the viewpoint of making the densities of the openings 30A of the upper surface 28A and the lower surface 28B of the porous film 28 uniform, it is preferable that the plurality of openings 30A are regularly arranged. A regular arrangement is an arrangement in which the coefficient of variation of the surface area of the parallel hexagonal or parallelogram unit of the above arrangement is, for example, 10% or less. Some of the openings 30A may be missing or the openings 30A may be out of alignment. However, it is preferable that the openings 30A are arranged in all directions without continuous gaps. The "coefficient of variation" is a value obtained by dividing the standard deviation of a predetermined population by the average, and is an index expressing the variance of the population as a percentage.

As shown in FIG. 3, each through hole 30 of the porous membrane 28 has a spherical missing shape, which is a shape in which the upper end and the lower end of the sphere are cut off. The through holes 30 adjacent to each other communicate with each other through the communication holes 34 inside the porous membrane 28.

Each through hole 30 preferably communicates with all adjacent through holes 30. When the openings 30A of the plurality of through holes 30 are arranged in a honeycomb shape as in this exemplary embodiment, each through hole 30 is connected to six adjacent through holes 30 through the six communication holes 34, respectively. Communicate. The through hole 30 may have a tubular shape, a columnar shape, a polygonal columnar shape, or the like, and the communication hole 34 may be a tubular void connecting adjacent through holes 30 to each other.

The opening diameter D of each opening 30A of the through hole 30 is, for example, a size through which red blood cells in the blood can pass. Specifically, the average opening diameter is preferably 1 μm to 20 μm, more preferably 3 μm to 10 μm. When the average opening diameter is set to 1 μm or more, the through hole 30 becomes a size through which red blood cells can pass, and when the average opening diameter is set to 20 μm or less, the vascular endothelial cell layer 36 and the cell layer 38 are retained on the main surface of the porous membrane 28. Will be able to.

Here, the "opening diameter D" is the long axis of the opening 30A, and the "average opening diameter" is the calculated average of the opening diameters D measured for 10 or more arbitrarily selected openings 30A. The "long axis" means the longest distance between two arbitrarily selected points on the contour of the aperture. However, when a direction is specified, the "long axis" means the longest distance between two arbitrarily selected points along that direction.

The opening ratio of the opening 30A of the through hole 30 is preferably 30% to 70%, more preferably 40% to 60%. When the aperture ratio is set to 30% or more, it is possible to suppress the movement of erythrocytes from being hindered by the porous membrane 28, and when the aperture ratio is set to 70% or less, the strength required for the porous membrane 28 can be achieved.

Here, the "aperture ratio" indicates the ratio of S2 to S1 as a percentage, and S1 assumes that the main surface of the porous membrane 28 is smooth (that is, the porous membrane 28 does not have an opening 30A). Below), the unit of surface area of the porous membrane 28, S2 indicates the total surface area of the openings 30A provided per unit surface, where equal units of measurement are used for S1 and S2.

The film thickness T of the porous membrane 28 is preferably half or less of the average opening diameter of the opening 30A of the through hole 30. Specifically, the thickness T is preferably 0.5 μm to 10 μm, and more preferably 1 μm to 10 μm. When the film thickness T of the porous membrane 28 is set to a thickness of half or less of the average opening diameter of the through hole 30, it is possible to prevent the porous membrane 28 from hindering the movement of red blood cells.

Further, since the porous membrane 28 is a scaffold on which cells adhere and grow, the cell-cell interaction between the cells on one surface of the porous membrane 28 and the cells on the other surface of the porous membrane 28, that is, a fluid factor. At least one of information transmission through, or cell-cell contact, becomes more active as the opening ratio on the porous membrane 28 increases and as the film thickness of the porous membrane 28 decreases. The more active the cell-cell interactions between cell cultures to provide the vascular endothelial cell layer 36 and the cell layer 38 on the main surface of the porous membrane 28, the more the vascular model having functionality similar to that of in vivo tissue. Can be generated well.

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 is, 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 set to 50% or more, it is possible to prevent the perforated membrane 28 from hindering the movement of red blood cells. If the porosity is too large, the strength of the porous membrane 28 will be insufficient with respect to the strength required for that purpose, so the porosity is preferably 95% or less.

Here, the "porosity" is the ratio of V2 to VI expressed as a percentage, and V1 is open to the porous film 28 under the assumption that the main surface of the porous film 28 is smooth (that is, the opening in the porous film 28). (Assuming there is no 30A), the unit of bulk of the porous film 28 is shown, where V2 is the sum of the bulks of the through holes 30 and the communication holes 34 provided per unit bulk, where V1 and V2 Equal units of measure are used.

The tensile elongation at break of the porous membrane 28 is preferably 50% or more, more preferably 100%, and even more preferably 200% or more. The stress required for 10% elongation of the porous membrane 28 is preferably 1000 gf / mm ² or less. The material becomes more flexible by increasing the tensile elongation at break and reducing the stress required for 10% elongation. Therefore, the perforated membrane 28 can be bent, stretched, and compressed, allowing the blood vessel model 10 to be closer to the actual blood vessel.

Here, the "tensile fracture elongation" can be evaluated by measuring the tensile elongation at break of the porous membrane 28 by the method specified in JIS K 6251: 2010. "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% by the method specified in JIS K 6251: 2010.

Examples of the method for producing the porous film 28 in which the through hole 30 is formed include a nanoprint method, a dew condensation method, an etching method, a sandblasting step, or a press molding step. The nanoprint method creates through holes 30 by pouring the material constituting the porous membrane 28 into a mold having protrusions and recesses, or by pressurizing such a mold against the material constituting the porous membrane 28. The method. The dew condensation method is a method of inducing condensation on the surface of a material constituting the porous membrane 28 and molding water droplets to form a through hole 30.

Compared with other methods, the dew condensation method can make the film thickness of the porous membrane 28 thinner, increase the porosity and aperture ratio of the opening 30A, and make the communication holes 34 in the porous membrane 28. Can be provided. Therefore, in this exemplary embodiment, the porous membrane 28 is manufactured using the condensation method. The dew condensation method is described in detail in, for example, Japanese Patent No. 4945281, Japanese Patent No. 5422230, Japanese Patent No. 5405374, and Japanese Patent Application Laid-Open No. 2011-74140.

Next, a case where drug toxicity evaluation is performed using the blood vessel model 10 of this exemplary embodiment will be described as an example. When conducting a drug toxicity test, first, the upper flow path member 12 and the lower flow path member 14 are joined to the porous membrane 28 in a state of being sandwiched between them, and as shown in FIG. 2, the upper micro flow path 16 and the lower micro flow path 16 and the lower micro are joined. A blood vessel model 10 including a flow path 22 is generated. The vascular endothelial cell layer 36 and the cell layer 38 are provided on the main surface of the porous membrane 28.

Then, using a pump, a blood diluent containing a drug is passed through a tube (not shown) and a through hole 20A into the upper microchannel 16 and passed through the inside of the upper microchannel 16 and then through hole 20B. And through a tube (not shown) and drained from the vascular model 10.

On the other hand, using a pump, the culture solution or physiological saline solution is passed through a tube (not shown) and the through hole 26A into the lower microchannel 22, passed through the inside of the lower microchannel 22, and then through hole 26B. And through a tube (not shown) and drained from the vascular model 10. The pressure of the upper microchannel 16 through which the blood diluent flows is higher than the pressure of the lower microchannel 22 through which the culture solution or physiological saline solution flows.

At the start of the toxicity test, as shown in FIG. 3, the entire upper surface 28A and the entire lower surface 28B of the porous membrane 28 are covered with the vascular endothelial cell layer 36 and the cell layer 38, respectively. Therefore, red blood cells in the blood do not leak into the lower microchannel 22 which can pass through the porous membrane 28.

However, after a certain period of time from the start of the toxicity test, the vascular endothelial cell layer 36 is damaged by the toxicity of the drug. In addition to the vascular endothelial cell layer 36, the cell layer 38 is also damaged by the drug. By measuring the number of red blood cells that have passed through the porous membrane 28 and flowed into the lower microchannel 22 by such a damaged portion, that is, by performing a bleeding evaluation, the vascular endothelial cell layer 36 and the cell layer 38 The level of drug-induced injury to the patient can be assessed.

Further, when the vascular endothelial cell layer 36 is damaged by the toxicity of the drug, the state of the cells constituting the cell layer 38 is changed by the cell-cell interaction between the vascular endothelial cell layer 36 and the cell layer 38, and as a result. , There is a possibility that a gap may be formed in the cell layer 38. By measuring the number of red blood cells that have passed through the gap and flowed into the lower microchannel 22, that is, by performing a bleeding evaluation, the level of drug-induced damage to the vascular endothelial cell layer 36 and the level of reaction of the cell layer 38 can be determined. Can be evaluated.

In this test, the cell-cell interaction between the vascular endothelial cell layer 36 and the cell layer 38 becomes more active as the aperture ratio on the porous membrane 28 increases and as the film thickness of the porous membrane 28 decreases. It can be done with high sensitivity.

Further, in the above toxicity test, a solution containing a drug and a tracer may be flowed through the upper microchannel 16 instead of the blood diluent. Examples of tracers include fluorescent-labeled chemicals, radioactive isotope-containing chemicals, dye compounds, and more specifically at least one selected from the group consisting of dextran, evance blue, sodium fluorescein and FITC microbeads. Is included. The fluorescent dye preferably has an excitation wavelength / fluorescence wavelength of 580 nm / 605 nm and is red.

The level of drug-induced damage to the vascular endothelial cell layer 36 and cell layer 38 is determined by measuring fluorescence intensity, radiation or chromaticity according to the type of tracer, quantifying the tracer, passing through the porous membrane 28 and from the upper microchannel 16 to the lower part. It can be evaluated by measuring the amount of tracer that has flowed into the microchannel 22.

In this exemplary embodiment, in the porous membrane 28 defining the upper microchannel 16 from the lower microchannel 22, the average opening diameter of the opening 30A of the through hole 30 is 1 μm to 20 μm, and the opening 30A of the through hole 30 The aperture ratio is configured to be 30% to 70%. Therefore, it is possible to suppress the movement of erythrocytes from being hindered by the porous membrane 28 during the bleeding evaluation in which the erythrocytes flowing through the upper microchannel 16 pass through the through hole 30 of the porous membrane 28 and move to the lower microchannel 22. it can.

Further, in this exemplary embodiment, the film thickness of the porous membrane 28 is configured to be half or less of the average opening diameter of the opening 30A of the through hole 30. Therefore, erythrocytes can more easily pass through the through hole 30 in the porous membrane 28 as compared with the case where the film thickness of the porous membrane 28 is larger than half of the average opening diameter of the opening 30A of the through hole 30. Therefore, this exemplary embodiment can further improve the accuracy of bleeding assessment.

Further, in this exemplary embodiment, the openings 30A of the through holes 30 arranged in a honeycomb shape are formed, and the through holes 30 in the porous membrane 28 communicate with each other 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, erythrocytes can more uniformly pass through the plurality of through holes 30 in the porous membrane 28. Therefore, this exemplary embodiment can further improve the accuracy of bleeding assessment.

Further, this exemplary embodiment is composed of a vascular endothelial cell layer 36 provided on the upper surface 28A of the porous membrane 28 and a cell layer 38 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. Further, the porous membrane 28 is made of a flexible material having a tensile elongation at break of 50% or more and a stress required for elongation of 10% of 1000 gf / mm ^{2 or less.} Thereby, in this 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 disclosure have been described. However, the present disclosure is not limited to the above, and can be embodied in various modifications other than the above without departing from the gist of the present disclosure.

For example, the opening 30A of the through hole 30 of the porous membrane 28 in the above exemplary embodiment has a circular shape in a plan view, but as shown in FIG. 5, the opening 50A of the through hole 50 of the porous membrane 48 is. It can have an elliptical shape in plan view. By forming the opening 50A of the through hole 50 in an elliptical shape, for example, a disc-shaped red blood cell can easily pass through the opening 50A of the through hole 50, and other cells in the blood can hardly pass through the opening 50A. Can be done.

An example of a method of forming the opening 50A of the opening 50A of the through hole 50 in an elliptical shape is that after forming a circular opening 30A as shown in FIG. 4 in the porous membrane 48, the porous membrane 48 is formed in one direction (horizontal direction in FIG. 4). ) Includes a method of stretching along. This method may form a plurality of elliptical openings 50A having major axial directions along the same direction (horizontal direction in FIG. 5).

Further, the elliptical opening 50A may be directly formed in the porous film 48 by press molding or the like without stretching the porous film 48. Further, as long as the shape of the opening 50A is a flat shape having a long axis and a short axis in a plan view, the shape of the opening 50A may be, for example, a flat polygon formed by extending a polygon.

In the porous membrane 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 membrane 28. However, as shown in FIG. 6, the porous film 58 has a porous region 62 in which the opening 60A of the through hole 60 is formed and a non-porous region 64 in which the opening 60A of the through hole 60 is not formed (2 in FIG. 6). The area indicated by the dotted line) can be provided.

Specifically, in the porous membrane 58, 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 the recess 24 constituting the lower microchannel 22 shown in FIG. The portions arranged in the vicinity of the inlet 24A and the vicinity of the outlet 24B of the above are configured as, for example, a non-porous region 64.

In general, the flow of liquids such as blood is easily disrupted at inlets 18A, 24A and outlets 18B, 24B. Therefore, by forming the porous membrane 58 near the inlets 18A and 24A and near the outlets 18B and 24B as the non-porous region 64, the flow of a liquid such as blood in the upper microchannel 16 and the lower microchannel 22 can be prevented. Can be adjusted. Therefore, the porous membrane 58 can further improve the accuracy of bleeding evaluation.

In the vascular model of the present disclosure, bleeding evaluation can be performed in a state where the movement of leaking substances such as erythrocytes to the outside of the blood vessel due to drug toxicity is suppressed by the porous membrane. Therefore, the blood vessel model of the present disclosure can be used as a blood vessel model capable of performing toxicity tests with high accuracy.

Examples of exemplary embodiments of the present disclosure will be described in detail below. The exemplary embodiments of the present disclosure should not be construed as being restricted by the examples exemplified below.

FIG. 7A shows a micrograph of the porous membrane of Example 1. In Example 1, the openings of the plurality of through holes are arranged in a honeycomb shape, the through holes are communicated through the communication holes, and a porous membrane formed of polycarbonate is used as in the porous membrane 28 of the above-exemplified embodiment. Was done. The average opening diameter of the opening of the porous membrane of Example 1 is 5 μm, the aperture ratio of the opening is 55%, the thickness of the porous membrane is 2.2 μm, and the coefficient of variation of the opening diameter of the opening is 3.5. %, The void ratio of the porous membrane was 75%, the tensile elongation at break was 250%, and the stress required for 10% elongation was 100 gf / mm ² .

The fine structure of the produced porous membrane was measured using a profile scanning laser microscope (product name: VK-X100, manufactured by Keyence, Japan). Observation was made using a magnification at which 50 or more openings appeared on a single screen. Based on the observed micrographs, each aperture diameter D was measured and image analysis was performed on the openings present on one screen to determine the average aperture diameter DAV and the coefficient of variation σD of the aperture diameter D. .. The coefficient of variation of the opening diameter (specified as a percentage) can be achieved using the calculation (σD / DAV) × 100.

The average aperture diameter and aperture ratio were achieved by subjecting the micrographs to binarization and image processing using 2D image analysis software WinROOF (manufactured by Mitani Shoji Co., Ltd.). The film thickness of the porous film is an average value of the thickness of the openings measured at 10 points using a profile scanning laser microscope.

The cross section of the porous membrane was observed using a scanning electron microscope (SEM, product name: SU8030, manufactured by Hitachi, Japan), and the diameter of a sphere equivalent to the through hole was calculated as the void ratio of the porous membrane. The porous membrane sample to be evaluated is sliced with a microtome (product name: FCS, manufactured by Reichert, Austria) to prepare a sample for cross-section observation, and the surface of the sample for cross-section observation is coated with an Os layer to a thickness of 6 nm, and the sample is prepared. It was observed by SEM using an acceleration voltage of 2 kV. The stress required for tensile elongation at break and 10% elongation of the porous membrane was measured using FUDOH RHEO METER RT-2002DD (manufactured by Rheotech Co., Ltd.).

FIG. 7B shows a micrograph of the porous membrane of Comparative Example 1. In Comparative Example 1, a conventional porous film made of polycarbonate having openings formed by a track etching method was used. The average opening diameter of the openings in the porous membrane of Comparative Example 1 was 5.7 μm, the aperture ratio of the openings was 12.4%, the film thickness of the porous membrane was 10.6 μm, and the opening diameter of the openings. The coefficient of variation was 35%, the void ratio of the porous membrane was 15%, the tensile elongation at break was 150%, and the stress required for 10% elongation was 5800 gf / mm ² .

The porous membrane is manufactured by adhering medical paper on both sides. The medical paper on one side of the porous membrane is removed using tweezers, and the side from which the medical paper has been removed is set downward on the lower flow path member. Next, the porous membrane is immersed in ethanol using a cotton swab to join the porous membrane and the lower flow path member.

Next, the medical paper on the other surface of the porous membrane is removed using tweezers, and the upper flow path member is laminated on the other surface of the porous membrane. The positions of the upper flow path member and the lower flow path member are aligned while being checked with a microscope, and the upper flow path member and the lower flow path member are joined. As a result, the blood vessel model of Example 1 and the blood vessel model of Comparative Example 1 were produced, respectively.

In Example 1 and Comparative Example 1, the porous membrane used for evaluating the permeability of the porous membrane to erythrocytes is a vascular endothelial cell layer 36 or smooth muscle cells provided on the main surface thereof, mesenchymal stem cells, and the like. It does not have a cell layer of cells selected from the group consisting of pericytes and fibroblasts.

In Example 2, cells are taken by taking the vascular model of Example 1, forming a rat vascular endothelial cell layer on the upper surface of the porous membrane, and forming a cell layer composed of rat smooth muscle cells on the lower surface of the porous membrane. A vascular model with layers was produced.

In Comparative Example 2, cells were taken by taking the vascular model of Comparative Example 1, forming a rat vascular endothelial cell layer on the upper surface of the porous membrane, and forming a cell layer composed of rat smooth muscle cells on the lower surface of the porous membrane. A vascular model with layers was produced.

In Example 2 and Comparative Example 2, rat vascular endothelial cells manufactured by Angio-Proteomie were used as rat vascular endothelial cells, and rat aortic smooth muscle cells manufactured by Lonza were used as rat smooth muscle cells. Lower microchannel, initial cell concentration was seeded at cell suspension 100μL of rat smooth muscle cells which is 3 × 10 ⁶ cells / ml. After one day incubation, cell concentration and seeded with cell suspensions 100μL of rat vascular endothelial cells is 3 × 10 ⁶ cells / ml in the upper microchannel. Two days after culturing, cell layer-attached blood vessel models of Example 2 and Comparative Example 2 were obtained.

A blood diluent having a red blood cell count of 3.7 × 10 ⁵ cells / ml was flowed through the upper microchannel of the vascular model produced in Example 1 and Comparative Example 1, and a saline solution was flowed through the lower microchannel. did. The fluid transfer rate of the blood diluent and the aqueous saline solution was set at 500 μL / min, the internal pressure of the upper microchannel was set at about 8.7 kPa, and the internal pressure of the lower microchannel was set at about 1.3 kPa. , Established parameters close to the actual blood flow and blood pressure conditions inside the blood vessel.

After a lapse of a predetermined time from the start of the fluid transfer, the internal lower microchannel, that is, the number of saline solution inside the red blood cells, providing a number of red blood cells in Example 1, 9.2 × 10 ⁴ cells / ml and provided the number of red blood cells 2.2 × 10 ⁴ cells / ml in the blood vessel model in Comparative example 1.

In this test, it was confirmed that both the porous membranes of Example 1 and Comparative Example 1 had permeability to erythrocytes under the same conditions as blood pressure. Further, it was confirmed that the porous membrane of Example 1 has higher erythrocyte permeability than the porous membrane of Comparative Example 1, and that the porous membrane of this exemplary embodiment can suppress the hindrance of the movement of erythrocytes.

The culture medium dilution containing 1.81 × 10 ⁶ beads / ml concentration of fluorescent beads relative tracer, flowing through the upper micro-channel of a cell layer adhered vessel model produced in Example 2 and Comparative Example 2, a fluorescent The bead-free culture medium was run through the lower microchannel. The fluorescent beads were labeled with a red fluorescent dye having a diameter of 4 μm, an excitation wavelength of 580 nm and a fluorescence wavelength of 605 nm. The fluid transfer rate of the culture medium diluent containing fluorescent beads and the culture medium not containing fluorescent beads was set to 500 μL / min, the internal pressure of the upper microchannel was set to about 8.7 kPa, and the inside of the lower microchannel. The pressure was set to about 1.3 kPa and parameters close to the actual blood flow and blood pressure conditions inside the blood vessel were established.

After a lapse of a predetermined time from the start of the fluid transfer, the internal lower microchannel, that is, the number of the culture medium in the fluorescent beads, fluorescent number beads of Example 2, 6.5 × 10 ⁴ beads / ml Provided, and in Comparative Example 2, the ^{number of fluorescent beads of 9.2 × 10 3} beads / ml was provided. A saline diluted solution containing fluorescent beads at 1.81 × 10 ⁶ beads / ml was flowed through the upper microchannel of the blood vessel model produced in Example 1 and Comparative Example 1, and the saline solution was flowed through the lower microchannel. Shed through. The fluid transfer rate was set to 500 μL / min. The number of fluorescent beads inside the lower microchannel ^{provided 1.7 × 10 5} beads / ml in Example 1 and 4.3 × 10 ⁴ beads / ml in Comparative Example 1. Provided. In this test, it was confirmed that forming cell layers on both sides of the porous membrane reduces the permeability of the porous membrane to fluorescent beads and imparts barrier properties to the porous membrane.

The cells produced in Example 2 and Comparative Example 2 by flowing the drug cytochalasin through the upper microchannel and the lower microchannel respectively at a concentration of 50 μg / ml and a flow rate of 0.7 μL / min for 1 day. The cell layers on both sides of the perforated membrane of the layered vascular model were exposed to the drug.

After drug exposure, a fluorescent bead permeability test was performed using the same method as the fluorescent bead permeability test for the cell layer-attached blood vessel model described above. After a lapse of a predetermined time from the start of the fluid transfer, the internal lower microchannel, that is, the number of the culture medium in the fluorescent beads, fluorescent number beads of Example 2, 1.7 × 10 ⁵ beads / ml Provided, and in Comparative Example 2, the ^{number of fluorescent beads of 6.7 × 10 3} beads / ml was provided.

In this test, it was confirmed that the fluorescent beads could pass through the porous membrane in the cell layer-attached blood vessel models of Example 2 and Comparative Example 2 after the cell layer was damaged by the drug. Further, the porous membrane of Example 2 has higher permeability to the fluorescent beads than the porous membrane of Comparative Example 2, and the porous membrane of this exemplary embodiment suppresses the movement of the fluorescent beads from being hindered. I was able to confirm that. Therefore, it was confirmed that the porous membrane of this exemplary embodiment can evaluate drug toxicity in a vascular model with high sensitivity.

In Example 3, similarly to Example 2, a cell layer-attached blood vessel model was produced from 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.

In Comparative Example 3, similarly to Comparative Example 2, a cell layer-attached blood vessel model was produced from 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.

The cells used in Example 3 and Comparative Example 3 were the same as the cells used in Example 2 and Comparative Example. To form a cell layer, the lower microchannel, initial cell concentration was seeded at cell suspension 100μL of rat smooth muscle cells which is 3 × 10 ⁶ cells / ml. One day after culturing, the upper microchannel was seeded with 100 μL of a cell suspension of rat vascular endothelial cells ^{having a cell concentration of 1 × 10 6 cells / ml, and cultured for 6 hours.} Next, each culture medium (rat EC medium / SMC medium) was flowed through each channel at a fluid transfer rate of 0.7 μL / min using a pump. Two days after culturing, cell layer-attached blood vessel models of Example 3 and Comparative Example 3 were obtained.

The lower microchannel of the cell layer-attached blood vessel model produced in Example 3 and Comparative Example 3 is closed and contains FITC-dextran (46945, manufactured by Sigma-Aldrich) at a concentration of 12.5 μg / 50 μl with respect to the tracer. The culture medium diluent was flowed through the upper microchannel. The fluid transfer rate of the culture medium diluent containing FITC-dextran was set to 7 μL / min.
Two minutes after flowing FITC-dextran through the channel, fluorescence in the microchannel was imaged using an inverted microscope (product name: EclipseTs2, manufactured by Nikon Corporation). Magnification: 4x, gain: 1600, and exposure time: 60ms were used for the imaging parameters. These results are shown in FIGS. 8A and 8B. No fluorescence was observed in the lower flow path in both Example 3 and Comparative Example 3. This indicates that FITC-dextran did not penetrate from the upper channel to the lower channel. In this test, it was confirmed that forming cell layers on both sides of the porous membrane suppresses FITC-dextran permeation and imparts barrier properties to the porous membrane.

By flowing the drug Phenoldpam through the upper microchannel at a concentration of 500 ng / ml and a flow rate of 0.7 μL / min for 1 day, the porous membrane of the cell layer-attached blood vessel model produced in Example 3 and Comparative Example 3 was prepared. The vascular endothelial cell layer was exposed 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-attached blood vessel model described above. These results are shown in FIGS. 9A and 9B. In the cell layer-attached blood vessel model of Example 3, fluorescence was observed over a wide range including the lower microchannel in addition to the upper microchannel. In the cell layer-attached blood vessel model of Comparative Example 3, the fluorescence observed in the lower microchannel was minimal. In this test, it was confirmed that FITC-dextran in both the cell layer-attached vascular models of Example 3 and Comparative Example 3 could pass through the porous membrane after the cell layer was damaged by the drug. Further, since the porous membrane of Example 3 was more permeable to FITC-dextran than the porous membrane of Comparative Example 3, the porous membrane of this exemplary embodiment does not interfere with the movement of FITC-dextran. I was able to confirm. Therefore, it was confirmed that the porous membrane of this exemplary embodiment can evaluate drug toxicity in a vascular model with high sensitivity.

In Example 4, the blood vessel model was manufactured by providing a 12 mm × 0.2 mm opening in the straight portion of the upper and lower flow paths of the blood vessel model of Example 1. The opening was formed by inserting a polypropylene reinforcing member having a 0.2 mm wide slit formed between the lower flow path member and the porous membrane. The reinforcing member has a thickness of 100 μm. 10A and 10B show micrographs of the porous membrane of Example 4.

In Example 5, collagen (5005-100ML, manufactured by AdvancedBioMatrix) was sprayed on the porous membrane of the blood vessel model of Example 1 at 60 ° C. for 15 minutes, and then the collagen was dried to form a thick coating, and then the porous membrane was formed. A cell layer-attached blood vessel model was produced by forming a rat vascular endothelial cell layer on the upper surface of the membrane and forming a rat smooth muscle cell layer on the lower surface of the porous membrane. FIG. 11 shows a micrograph of the porous membrane of Example 5.

In Example 6, the blood vessel model of Example 4 was taken, and a rat vascular endothelial cell layer was formed on the upper surface of the porous membrane to produce a blood vessel model to which a single cell layer was attached.

A diluted solution of culture medium containing 1.81 × 10 ⁶ beads / ml concentration of fluorescent beads was flowed through the upper microchannel of the single cell layer-attached vascular model produced in Example 6 to the tracer, and the fluorescent beads were flown. The culture medium containing no was flowed through the lower microchannel. The fluorescent beads were labeled with a red fluorescent dye having a diameter of 4 um, an excitation wavelength of 580 nm and a fluorescence wavelength of 605 nm. The fluid transfer rate of the culture medium diluent containing fluorescent beads and the culture medium not containing fluorescent beads was set to 500 μL / min, the internal pressure of the upper microchannel was set to about 8.7 kPa, and the inside of the lower microchannel. The pressure was set to about 1.3 kPa and parameters close to the actual blood flow and blood pressure conditions inside the blood vessel were established.

After a lapse of a predetermined time from the start of the fluid transfer, the internal lower microchannel, that is, the number of the culture medium in the fluorescent beads, fluorescent number beads 2.67 × 10 ⁴ beads / ml in Example 6 Provided. Flowing the saline diluent containing 1.81 × 10 ⁶ beads / ml of fluorescent beads through the upper flow passage of the vessel model of Example 4, and saline through the lower passage at 500 [mu] L / min fluid transfer rate We have provided a number of fluorescent beads 7.23 × 10 ⁵ beads / ml in example 4 to flow. In this test, it was confirmed that forming a cell layer on a single surface of the porous membrane reduces the permeability of the porous membrane to fluorescent beads and imparts barrier properties to the porous membrane.

In Example 7, the vascular model of Example 1 was taken, a human vascular endothelial cell layer derived from induced pluripotent stem cells was formed on the upper surface of the porous membrane, and human mesenchymal stem cells were formed on the lower surface of the porous membrane. , Generated a cell layer-attached blood vessel model.

In the eighth embodiment, the porous membrane formed of polycarbonate is similar to the porous membrane 28 of the above-exemplified embodiment in which the openings of the plurality of through holes are arranged in a honeycomb shape and the through holes are communicated through the communication holes. It was used. The average opening diameter of the opening of the porous membrane of Example 8 is 3 μm, the aperture ratio of the opening is 52%, the thickness of the porous membrane is 1.2 μm, and the coefficient of variation of the opening diameter of the opening is 5.0. The void ratio of the porous membrane was 80%.

In Example 9, the blood vessel model of Example 8 was taken, 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 to produce a cell layer-attached blood vessel model. ..

Claims (8)

A pair of flow path members facing each other, including facing surfaces on which each micro flow path is formed,
Includes a plurality of through-holes penetrating the porous film in the thickness direction, is arranged between the opposing surfaces of the pair of the channel members, said porous membrane defining between microchannel,
With
The porous membrane is provided with a vascular endothelial cell layer, and the vascular endothelial cell layer covers one surface facing one of the microchannels.
The average opening diameter of the through hole is 1 [mu] m ~ 20 [mu] m,
The aperture ratio of the through hole is 30% to 70%
The film thickness of the porous membrane is less than half the average opening diameter of the through hole.
Blood vessel model. The blood vessel model according to claim 1, wherein the film thickness of the porous membrane is 0.5 μm to 10 μm. The porous membrane is of formed inside, further comprising a communication hole for communicating the through hole with each other,
The through holes are arranged in a honeycomb shape, and 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 membrane is 50% or more.
The blood vessel model according to claim 1. Is provided on the other surface of said porous membrane opposite to the other one the microchannel of smooth muscle cells, mesenchymal stem cells, pericytes, and cells selected from the group consisting of fibroblasts The blood vessel model according to claim 1, further comprising a cell layer of. Tensile elongation at break of the porous membrane is not less than 50%,
The blood vessel model according to claim 1, wherein the stress required for 10% elongation of the porous membrane is 1000 gf / mm ^{2 or less.} The blood vessel model according to claim 1, wherein the through hole has a flat shape in a plan view and includes a long axis and a short axis. The porous membrane, the porous region and the through hole through hole is formed includes a non-porous region is not formed, vessel model according to claim 1. The blood vessel model of claim 1 is provided.
Flowing a blood diluent containing the drug in the microchannel facing the surface of the porous membrane vascular endothelial cell layer is provided,
Count the number of red blood cells to be leaked into the micro flow path opposite to the other surface of the porous membrane,
A method of evaluating bleeding using a blood diluent containing a drug.

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