Biopsy phantom device, blood vessel wall phantom device, and method for evaluating test substance

文档序号：1580327 发布日期：2020-01-31 浏览：24次中文

阅读说明：本技术 活组织模型装置、血管壁模型，血管壁模型装置及评价被检物质的方法 (Biopsy phantom device, blood vessel wall phantom device, and method for evaluating test substance ) 是由伊藤晃寿柿沼千早西野雅史美马伸治 C·M·内维尔 C·A·森德巴克于 2018-06-07 设计创作，主要内容包括：本发明提供一种活组织模型装置、血管壁模型及血管壁模型装置。所述活组织模型装置包括：第一液体隔室,储存液体组合物；第二液体隔室,储存液体组合物；及细胞层叠体,配置于第一液体隔室与第二液体隔室之间作为两个隔室之间的隔层。所述血管壁模型包括：多孔膜,具有蜂窝结构；血管内皮细胞层,配置于多孔膜的一表面；及平滑肌细胞层或间充质干细胞层,配置于多孔膜的另一表面。血管壁模型装置包括：第一液体隔室,储存液体组合物；第二液体隔室,储存液体组合物；及血管壁模型,配置于第一液体隔室与第二液体隔室之间作为两个隔室的隔层。本发明还提供一种这些模型或模型装置的应用。(The invention provides living tissue model devices, a blood vessel wall model and a blood vessel wall model device, wherein the living tissue model device comprises a th liquid compartment for storing a liquid composition, a second liquid compartment for storing the liquid composition and a cell laminated body arranged between a th liquid compartment and the second liquid compartment as a partition between the two compartments, the blood vessel wall model comprises a porous membrane with a honeycomb structure, a blood vessel endothelial cell layer arranged on the surface of the porous membrane and a smooth muscle cell layer or an mesenchymal stem cell layer arranged on the other surface of the porous membrane, the blood vessel wall model device comprises a th liquid compartment for storing the liquid composition, a second liquid compartment for storing the liquid composition and a blood vessel wall model arranged between a th liquid compartment and the second liquid compartment as a partition between the two compartments, and the invention also provides application of the models or model devices.)

1, a living tissue phantom device, comprising:

a liquid compartment for storing a liquid composition;

a second liquid compartment storing a liquid composition; and

a stack of cells disposed between the th fluid compartment and the second fluid compartment as a barrier between the th fluid compartment and the second fluid compartment,

the cell laminate comprises a porous membrane having a honeycomb structure, a cell layer which contains th cells and is disposed on the surface of the porous membrane, and a cell layer which contains a second cell different from the th cell and is disposed on the other surface of the porous membrane.

2. The living tissue phantom device according to claim 1, wherein,

the th cell and the second cell are two cells selected from the group consisting of a parenchymal cell, a stromal cell, a muscle cell, a fibroblast, a nerve cell, a glial cell, an endothelial cell, and an epithelial cell.

3. The living tissue phantom device according to claim 1, wherein,

the porous membrane material includes at least selected from the group consisting of polybutadiene, polystyrene, polycarbonate, polysulfone, polyurethane, polylactic acid-polyglycolic acid copolymer, polylactic acid-polycaprolactone copolymer, polyethylene terephthalate, poly (glycerol sebacate), polyacrylate, polymethacrylate, polyacrylamide, polyethylene naphthalate, polyethylene glycol succinate, polybutylene succinate, polycaprolactone, polyamide, polyimide, polysiloxane derivative, and cellulose triacetate.

4. The living tissue phantom device according to claim 1, wherein,

each surface of the porous membrane is coated with at least selected from the group consisting of fibronectin, collagen, laminin, vitronectin, gelatin, perlecan, entactin, proteoglycan, osteopontin, tenascin, nephronexin, basement membrane matrix, recombinant peptides, and polylysine.

5. The living tissue phantom device according to claim 1, wherein,

the average diameter of the openings of the through holes on the porous membrane is 1-20 μm, and the opening ratio of the porous membrane is 30-70%.

6, A method for evaluating a test substance using the living tissue phantom apparatus of claim 1, comprising:

a step of adding a test substance to at least of the th liquid compartment or the second liquid compartment, and

(i) at least steps of (i) quantifying at least chemical substances contained in the th liquid compartment or cells contained in the th liquid compartment, or (ii) quantifying at least chemical substances contained in the second liquid compartment or cells contained in the second liquid compartment.

7. The method for evaluating a test substance according to claim 6,

the step (i) comprises quantifying at least of the mirnas contained in the th liquid compartment, the proteins contained in the th liquid compartment, or the transcription factors contained in the th liquid compartment, and the step (ii) comprises quantifying at least of the mirnas contained in the second liquid compartment, the proteins contained in the second liquid compartment, or the transcription factors contained in the second liquid compartment.

8. The method for evaluating a test substance according to claim 6, further comprising:

a step of adding a tracer to the liquid compartment to which the test substance has been added, wherein the step of measuring the amount of tracer leaking from the liquid compartment to which the tracer has been added to another liquid compartment constitutes step (i) or (ii).

The blood vessel wall models of , comprising:

a porous membrane having a honeycomb structure;

a vascular endothelial cell layer disposed on the surface of the porous membrane, and

a smooth muscle cell layer or a mesenchymal stem cell layer disposed on the other surface of the porous membrane.

10. The blood vessel wall model of claim 9,

in the blood vessel wall model, the permeability of FITC-dextran 70 from the vascular endothelial cell layer side to the smooth muscle cell layer side or the mesenchymal stem cell layer side is 0% to 10% of the permeability of FITC-dextran 70 from the surface of the porous membrane to the other surface of the porous membrane.

11. The blood vessel wall model of claim 9,

12. The blood vessel wall model of claim 9,

each surface of the porous membrane is coated with at least selected from the group consisting of fibronectin, collagen, laminin, vitronectin, gelatin, perlecan, entactin, proteoglycan, osteopontin, tenascin, nephronectin, basement membrane matrix, recombinant peptide, and polylysine.

13. The blood vessel wall model of claim 9,

the average diameter of the openings of the through holes of the porous membrane is 1-20 μm, and the opening ratio of the porous membrane is 30-70%.

14, a vessel wall modeling apparatus, comprising:

a liquid compartment for storing a liquid composition;

a second liquid compartment storing a liquid composition; and

the blood vessel wall phantom of claim 9, disposed between the th liquid compartment and the second liquid compartment as a barrier between the th liquid compartment and the second liquid compartment.

15, a method for evaluating a test substance using the blood vessel wall model device of claim 14, comprising:

a step of adding a test substance to at least of the th liquid compartment or the second liquid compartment, and

16. The method for evaluating a test substance according to claim 15,

17. The method for evaluating a test substance according to claim 15,

of the or second liquid compartments are liquid compartments storing blood, a liquid composition containing red blood cells or a liquid composition mimicking blood and containing at least kinds of liquid compositions selected from dextran, evans blue, fluorescein sodium salt and FITC-microspheres, the step of adding a test substance to at least of the or second liquid compartments includes the step of adding a test substance to liquid compartments storing blood, a liquid composition containing red blood cells or a liquid composition mimicking blood and containing at least kinds of liquid compositions selected from dextran, evans blue, fluorescein sodium salt and FITC-microspheres, and the amount of red blood cells leaking from the liquid compartment to which the test substance has been added to the another liquid compartment is measured, the amount of hemoglobin leaking from the liquid compartment to which the test substance has been added to the another liquid compartment or the amount of dextran salt leaking from the liquid compartment to which the test substance has been added to the another 362 liquid compartment is selected from dextran, evans sodium salt and FITC-4934 (73ii) of the step of adding at least kinds of liquid compositions selected from dextran, evans blue, fluorescein sodium salt and FITC-microspheres).

Technical Field

The present invention relates to a living tissue model device, a blood vessel wall model device, and a method for evaluating a test substance.

Background

Japanese patent No. 5,113,332 discloses blood-brain barrier in vitro models having a structure in which a filter device called a "cell culture insert" is inserted into a culture dish, and having a structure in which a brain capillary endothelial cell layer is disposed on the upper surface of the filter of the cell culture insert, a brain pericyte layer is disposed on the lower surface of the filter of the cell culture insert, and an astrocyte layer is disposed on the bottom surface of the culture dish, and a method for evaluating drugs using the same.

In this blood brain barrier in vitro model, the filter portion of the cell culture insert was a laminate of a brain capillary endothelial cell layer, a track-etched (TE) membrane, and a pericephalic cell layer obtained by culturing pericephalic cells on the surface of the TE membrane, followed by culturing brain capillary endothelial cells on the other surface of the TE membrane.

Japanese patent No. 5,113,332 discloses methods of using the blood brain barrier in vitro model, which includes a step of adding a drug to the inside of a cell culture insert (a liquid compartment on the side where a brain capillary endothelial cell layer is arranged), a step of measuring the amount of the drug leaking to the outside of the cell culture insert (a liquid compartment on the side where a pericyte layer is arranged), and an evaluation of the ability of the drug to pass through the blood brain barrier.

Disclosure of Invention

Technical problem to be solved by the invention

In order to obtain a living tissue model device for evaluating a drug or a disease state, which can replace an animal test, it is necessary to construct a cellular tissue having a structure and a function similar to those of a tissue in an organism. From the viewpoint of a cellular tissue having a structure and a function similar to those of a tissue in a living body, it is preferable to obtain a cell laminate by culturing cells on both sides of a porous membrane having an opening ratio higher than a TE membrane (the TE membrane generally has an opening ratio of about 2% to about 20%) which serves as a cell culture scaffold, and to apply the cell laminate to a living tissue model device.

As ｃA cell culture scaffold, there are known ｃA honeycomb structure membrane disclosed in Japanese patent application laid-open (JP-A) No. 2002-335949 and ｃA honeycomb membrane disclosed in Japanese patent application laid-open (JP-A) No. 2007-006987 JP-A No. 2002-335949 discloses cell laminates obtained by culturing the same kind of cells (stem cells or cardiomyocytes) on both sides of the honeycomb structure membrane, and JP-A No. 2007-006987 discloses cell sheets for transplantation for skin regeneration obtained by culturing fibroblasts on the surface of the honeycomb membrane and then culturing epithelial keratinocytes on the other surface of the honeycomb membrane.

Embodiments according to the present invention have been made in view of the above circumstances.

The invention aims to provide novel living tissue model devices, novel blood vessel wall models, novel blood vessel wall model devices and application thereof, which are the problems to be solved by the invention.

Means for solving the technical problem

Specific methods for solving the above problems include the following aspects.

[A1] a living tissue model device, comprising:

a liquid compartment for storing a liquid composition;

a second liquid compartment storing a liquid composition; and

a stack of cells disposed between the th fluid compartment and the second fluid compartment as a barrier between the th fluid compartment and the second fluid compartment,

[A2] The living tissue phantom device according to [ A1], wherein,

the th cell and the second cell are two cells selected from the group consisting of parenchymal cells, stromal cells, muscle cells, fibroblasts, nerve cells, glial cells, endothelial cells, and epithelial cells.

[A3] The living tissue model device according to [ A1] or [ A2], wherein,

the material of the porous membrane includes at least selected from the group consisting of polybutadiene, polystyrene, polycarbonate, polysulfone, polyurethane, polylactic acid-polyglycolic acid copolymer, polylactic acid-polycaprolactone copolymer, polyethylene terephthalate, poly (glycerol sebacate), polyacrylate, polymethacrylate, polyacrylamide, polyethylene naphthalate, polyethylene glycol succinate, polybutylene succinate, polycaprolactone, polyamide, polyimide, polysiloxane derivative, and cellulose triacetate.

[A4] The living tissue model device according to of any of [ A1] to [ A3], wherein,

[A5] The living tissue model device according to of any of [ A1] to [ A4], wherein,

the average diameter of the openings of the through holes on the porous membrane is 1-20 μm, and the opening ratio of the porous membrane is 30-70%.

[A6] A method for evaluating a test substance using the living tissue model device of any of [ A1] to [ A5], comprising:

at least steps of adding a test substance to the th liquid compartment or the second liquid compartment, and

[A7] The method for evaluating a test substance according to [ A6], wherein,

[A8] The method for evaluating a test substance according to [ A6], further comprising:

[B1] A blood vessel wall model, comprising:

a porous membrane having a honeycomb structure;

a vascular endothelial cell layer disposed on the surface of the porous membrane, and

and a smooth muscle cell layer disposed on the surface of the porous membrane .

[B2] The blood vessel wall model according to [ B1], wherein,

in the blood vessel wall model, the FITC-glucan 70 permeability from the vascular endothelial cell layer side to the smooth muscle cell layer side is 0% to 10% of the FITC-glucan 70 permeability from the surface of the porous membrane to the other surface of the porous membrane.

[B3] A blood vessel wall model, comprising:

a porous membrane having a honeycomb structure;

a vascular endothelial cell layer disposed on the surface of the porous membrane, and

and a mesenchymal stem cell layer disposed on the surface of the another of the porous membrane.

[B4] The blood vessel wall model according to [ B3], wherein,

in the blood vessel wall model, the FITC-glucan 70 permeability from the vascular endothelial cell layer side to the mesenchymal stem cell layer side is 0% to 10% of the FITC-glucan 70 permeability from the surface of the porous membrane to the other surface of the porous membrane.

[B5] The blood vessel wall model of any of [ B1] to [ B4], wherein,

[B6] The blood vessel wall model of any of [ B1] to [ B5], wherein,

[B7] The blood vessel wall model of any of [ B1] to [ B6], wherein,

the average diameter of the openings of the through holes of the porous membrane is 1-20 μm, and the opening ratio of the porous membrane is 30-70%.

[C1] A blood vessel wall modeling apparatus, comprising:

th liquid compartment for storing a liquid composition, a second liquid compartment for storing a liquid composition, and of [ B1] to [ B7], disposed between the th liquid compartment and the second liquid compartment as a barrier between the th liquid compartment and the second liquid compartment.

[C2] A method for evaluating a test substance using the blood vessel wall model device described in [ C1], comprising:

at least steps of adding a test substance to the th liquid compartment or the second liquid compartment, and

[C3] The method for evaluating a test substance according to [ C2], wherein,

[C4] The method for evaluating a test substance according to [ C2], wherein,

of the or the second liquid compartments are liquid compartments storing blood, a liquid composition containing red blood cells or a liquid mimetic of blood and containing at least 0 liquid compositions selected from the group consisting of dextran, evans blue, sodium fluorescein salt and FITC-microspheres, the process of adding a test substance to at least of the or the second liquid compartments includes the process of adding a test substance to liquid compartments storing blood, a liquid composition containing red blood cells or a liquid mimetic of blood and containing at least liquid compositions selected from the group consisting of dextran, evans blue, sodium fluorescein salt and FITC-microspheres, and the amount of red blood cells leaking from the liquid compartment to which the test substance has been added to the other liquid compartment is measured, the amount of hemoglobin leaking from the liquid compartment to which the test substance has been added to the other liquid compartment or the amount of hemoglobin leaking from the liquid compartment to which the test substance has been added to the other liquid compartment includes at least one of the processes of evans blue, sodium fluorescein salt, and FITC- liquid compositions (processes) including at least one of the processes of dextran, evans blue, FITC-microspheres ().

[D1] A method of producing a cell laminate having cell layers on both sides of a porous membrane, the method comprising using a container having a bottom and a sidewall portion standing from an edge of the bottom, the porous membrane, and a holding member configured to hold the porous membrane so that the porous membrane faces an inner bottom surface of the container and is held at a position not in contact with the inner bottom surface, the method comprising:

culturing th cells in a liquid culture medium in contact with the inner bottom surface of the container and the surface of the porous membrane, the porous membrane being held by a holding member at a position not in contact with the inner bottom surface of the container so as to face the inner bottom surface, and the bottom of the container being located at the upper side and the porous membrane being located at the lower side in the direction of gravity, and

and culturing th cells on the lower surface of the porous membrane and culturing the second cells on the upper surface of the porous membrane in a state where the porous membrane is held by the holding member at a position not in contact with the inner bottom surface of the container so as to face the inner bottom surface, and the bottom of the container is positioned on the upper side and the porous membrane is positioned on the lower side in the direction of gravity.

Effects of the invention

According to the present invention, novel living tissue phantom devices, novel blood vessel wall phantoms, novel blood vessel wall phantom devices and uses thereof are provided.

Drawings

FIG. 1 is a schematic cross-sectional view of examples of a living tissue model device.

FIG. 2 is a schematic partial cross-sectional view showing examples of cell laminates in the biopsy device.

Fig. 3A is a perspective view of examples of porous films having a honeycomb structure.

Fig. 3B is a plan view of the porous membrane shown in fig. 3A as viewed from above.

Fig. 3C is a sectional view showing the porous membrane taken along line C-C in fig. 3B.

Fig. 4A is a perspective view of examples showing the holding member.

FIG. 4B is a perspective view showing a state in which the holding member shown in FIG. 4A is disposed in the culture vessel.

FIG. 5 is a schematic view of examples showing a method for producing a cell laminate.

FIG. 6 is a schematic view of examples showing a method for producing a cell laminate.

Fig. 7 is a photomicrograph of the porous film used in example 1.

FIG. 8 is an immunofluorescence image of a cell layer formed on any surfaces of the porous membrane in example 1.

FIG. 9 is a graph showing the relative fluorescence intensity of FITC-dextran 70.

FIG. 10 is a graph showing the relative fluorescence intensity of FITC-dextran 70.

FIG. 11 is an immunofluorescence image of a cell layer formed on any surfaces of the porous membrane in example 2.

FIG. 12 is an immunofluorescence image of the cell stack of example 2.

Detailed Description

Hereinafter, embodiments of the present invention will be described. The description and examples provided below illustrate exemplary embodiments without limiting the scope of the invention. The mechanism of action described in the present invention includes speculation, and the scope of the present invention is not limited to this speculation.

In the present invention, each numerical range represented by "to" includes ranges in which the numerical values before and after "to" are respectively a lower limit value and an upper limit value.

When two or more substances each corresponding to a specific component in the composition are present, the amount of the specific component described in the composition of the present invention means the total amount of the two or more substances present in the composition unless otherwise specified.

< living tissue model device and blood vessel wall model device >

The living tissue phantom apparatus according to the present invention comprises:

a liquid compartment for storing a liquid composition;

a second liquid compartment storing a liquid composition; and

a stack of cells disposed between the th fluid compartment and the second fluid compartment as a barrier between the th fluid compartment and the second fluid compartment,

the cell stack in the living tissue phantom device according to the present invention comprises:

a porous membrane having a honeycomb structure;

a cell layer containing th cells and disposed on the surface of the porous membrane having a honeycomb structure, and

a cell layer comprising a second cell different from the th cell and disposed on the other surface of the porous membrane having a honeycomb structure.

Hereinafter, the porous membrane having a honeycomb structure is also referred to as a "honeycomb membrane".

In the biopsy model device according to the invention the cell stack is arranged with cell layer facing the th liquid compartment and cell layer facing the second liquid compartment.

In the living tissue phantom device according to the present invention, the liquid composition stored in the th liquid compartment and the liquid composition stored in the second liquid compartment may have the same composition or different compositions from each other.

A living tissue phantom device 500, which is an example of a living tissue phantom device according to the present invention, is shown in FIG. 1, FIG. 1 is a schematic cross-sectional view of the living tissue phantom device 500, in FIG. 1, the dimensions of the various components are conceptual dimensions, and the relative relationship between the dimensions of the components is not limited thereto, the living tissue phantom device 500 includes a th fluid compartment 410, a second fluid compartment 420, and a cell stack 300, each of the th fluid compartment 410 and the second fluid compartment 420 stores a fluid composition, the fluid composition stored in the th fluid compartment 410 and the fluid composition stored in the second fluid compartment 420 may have the same composition or different compositions from each other, and the cell stack 300 is a barrier portion between the th fluid compartment 410 and the second fluid compartment 420.

The living tissue model device 500 shown in FIG. 1 has a structure in which a cell culture insert is disposed in a culture container, and the living tissue model device of this structure includes a container having a bottom and a side wall portion standing from the edge of the bottom, a cell culture insert disposed in the container, and a cell culture insert comprising a cell laminate, and is constituted by a culture container and a culture device in which the culture container and the cell culture insert are bodies (for example, a structure shown in FIG. 4B described below) according to a production method described below, and a cell culture insert obtained after producing the cell laminate is described below, and this structure is referred to as a "cell culture insert type device" as an example , and when the structure of the cell culture insert type device is described with reference to FIG. 4B, a space defined by the hollow cylindrical portion 42 of the holding member 40 and the honeycomb membrane 20 corresponds to the -th liquid compartment 410 shown in FIG. 1, and a space defined by the bottom 62 of the culture container 60, the side wall portion 64 of the culture container 60, the hollow cylindrical portion 42 of the holding member 40, and the honeycomb membrane 20 corresponds to the second liquid compartment 420 shown in FIG. 1.

examples of living tissue phantom devices according to the invention are vessel wall phantom devices the vessel wall phantom device according to the invention comprises:

a liquid compartment for storing a liquid composition;

a second liquid compartment storing a liquid composition; and

a vessel wall phantom disposed between the th liquid compartment and the second liquid compartment as a barrier between the th liquid compartment and the second liquid compartment,

the vascular wall model in the vascular wall model device according to the present invention includes a honeycomb membrane, a vascular endothelial cell layer disposed on a surface of the honeycomb membrane, and a smooth muscle cell layer or a mesenchymal stem cell layer disposed on another surface of the honeycomb membrane.

In the blood vessel wall model device according to the present invention, the liquid composition stored in the th liquid compartment and the liquid composition stored in the second liquid compartment may have the same composition or different compositions from each other, the liquid composition preferably has a composition composed in such a manner as to maintain the vascular endothelial cells and smooth muscle cells/mesenchymal stem cells in a living state, examples of the liquid composition include phosphate buffered saline, physiological saline, a basal medium for mammalian cells, blood, a liquid composition containing erythrocytes, and a liquid composition mimicking blood and containing at least species selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres.

cases of the structure of the living tissue phantom device according to the present invention are the structures of the cell laminate 300 which is the blood vessel wall model of the living tissue phantom device 500 shown in FIG. 1. cases of the structure of the living tissue phantom device according to the present invention include the above-described cell culture insert type device.

Hereinafter, a cell laminate in the living tissue phantom device according to the present invention and a vascular wall phantom in the vascular wall phantom device according to the present invention will be described.

[ cell laminate and vascular wall model ]

The cell laminate in the living tissue model device according to the present invention includes a honeycomb membrane, a cell layer including th cells and disposed on the surface of the honeycomb membrane, and a cell layer including a second cell different from the th cell and disposed on the other surface of the honeycomb membrane, and the number of the cell layers disposed on each surface of the honeycomb membrane may be 1 layer, 2 layers or more.

An example of a cell laminate 300 as a cell laminate in a living tissue phantom device according to the present invention is shown in fig. 2 is a schematic partial sectional view of the cell laminate 300 fig. 2, the dimensions of the respective members are conceptual dimensions, and the relative relationship between the dimensions of the members is not limited thereto.

The cell laminate 300 includes a honeycomb membrane 200, a cell layer 110 including th cells, and a cell layer 120 including second cells, the cell layer 110 including th cells is disposed on a main surface of the honeycomb membrane 200, and the cell layer 120 including second cells is disposed on another main surface of the honeycomb membrane 200.

[ Honeycomb film ]

The honeycomb membrane in the cell laminate according to the present invention is used as a scaffold for cell adhesion and proliferation in the production of the cell laminate. More specifically, cells proliferate on both sides of the honeycomb membrane to form cell layers on both sides, thereby providing the cell laminate according to the present invention.

The honeycomb structure of the present invention is a structure in which a plurality of through-holes are formed by being partitioned by partition walls. In the honeycomb membrane in the cell laminate according to the present invention, the through-holes of the honeycomb structure are opened at the main surface of the honeycomb membrane. The honeycomb membrane in the cell laminate according to the present invention may be a membrane having a structure in which a plurality of honeycomb structures are laminated in layers.

The shape of the through-holes is, for example, a truncated sphere, a cylinder, or a polygonal column in which portions of the spheres are absent, and through-holes of various shapes may be present at the same time.

In the honeycomb film, it is preferable that the through-holes are regularly arranged from the viewpoint of improving the uniformity of the cell layer arranged on the honeycomb film. The regular arrangement may include a break or an alternation. However, the regular arrangement preferably includes a continuous repetition without discontinuity in all directions.

Hereinafter, an example of the honeycomb film will be described with reference to the drawings. In the drawings, the same or equivalent elements or portions are denoted by the same reference numerals. In the following description, the longer diameter indicates the longest distance between any two points on the contour line, or indicates the longest distance between any two points in a specified direction when the direction is specified.

Fig. 3A to 3C show a honeycomb film 20 as an example of the honeycomb film, fig. 3A is a perspective view of the honeycomb film 20, fig. 3B is a plan view of the honeycomb film 20 shown in fig. 3A as viewed from above, and fig. 3C is a cross-sectional view of a porous film taken along the line C-C in fig. 3B.

The through-holes 22 are arranged in all regions on the main surface of the honeycomb film 20. However, when there is a region of the honeycomb membrane 20 that cannot be contacted with cells, it is not necessary to provide the through-holes 22 in this region. In the honeycomb film 20, adjacent through-holes 22 are separated from each other by partition walls 24.

In the arrangement of the through-holes 22, units are represented by a hexagonal shape (preferably a regular hexagonal shape) or the like whose opposite sides are parallel to each other, and the center of the opening is located at the intersection of the vertex and the diagonal line of the shape.

The shape of the through-holes 22 is, for example, a truncated sphere, a cylinder, or a polygonal column which is missing part of the sphere, the opening shape of the through-holes 22 is, for example, a circle, an ellipse, or a polygon, and in the honeycomb structure, the adjacent through-holes 22 can communicate with each other through the communication holes inside the honeycomb film 20.

The dimensions of the honeycomb film 20 will be described below.

The pitch P1 of the through holes 22 is the distance between the centers of adjacent openings. The pitch P1 is preferably adjusted according to the size of cells included in the cell layer disposed on the honeycomb membrane 20. The pitch P1 is, for example, 1 μm to 50 μm.

The opening diameter Da is a longer diameter of the opening of the through-hole 22, the opening diameter Da is preferably a size that allows cells contained in the cell layer to be held on the honeycomb membrane 20, the opening diameter Da is, for example, 10% to 150% of a longer diameter (for example, 10 μm to 50 μm) of cells contained in the cell layer, when the blood vessel wall model is constructed for performing the erythrocyte leakage test, the opening diameter Da is preferably a size that allows erythrocytes to pass through, the opening diameter Da is preferably not excessively large from the viewpoint of intercellular contact between the cells on the surface and the cells on the surface, the opening diameter Da is preferably not excessively small from the viewpoint of allowing the cells contained in the cell layer to be held on the honeycomb membrane 20, the opening diameter Da is preferably 1 μm to 20 μm, more preferably 2 μm to 10 μm, further is preferably 3 μm to 5 μm, and similarly, the average value of the opening diameter Da is preferably 1 μm to 20 μm, more preferably 2 μm to 10 μm, and further preferably 3 μm to 5 μm.

The coefficient of variation of the opening diameter Da is preferably 20% or less, and more preferably a smaller coefficient of variation. A smaller coefficient of variation of the opening diameter Da provides higher uniformity of the cell layer disposed on the honeycomb membrane 20. The coefficient of variation is a value obtained by dividing the standard deviation of the set by the arithmetic mean of the set, and is an index of the degree of variation within the set. In the present invention, the coefficient of variation is expressed as%.

The width W of the partition wall 24 is the width of the partition wall 24 measured as the shortest distance between adjacent openings. The width W is preferably a width that allows the cells contained in the cell layer to remain on the honeycomb membrane 20.

The opening ratio of the honeycomb film 20 is preferably 30% to 70%, more preferably 35% to 65%, and further preferably 40% to 60% in the step from the viewpoint of the substance permeability and the strength of the honeycomb film, the opening ratio of the honeycomb film 20 is the ratio of the total area of the openings to the main surface area (including the area of the openings) in a plan view, and the opening ratios on surfaces and on surfaces are calculated, respectively.

From the viewpoint of cell-cell contact between the cell on the surface of and the cell on the surface of , the thickness of the honeycomb membrane 20 is preferably not excessively thick, and from the viewpoint of the strength of the honeycomb membrane 20, the thickness of the honeycomb membrane 20 is preferably not excessively thin, and from these viewpoints, the thickness of the honeycomb membrane 20 is preferably 0.5 μm to 40 μm, more preferably 1 μm to 20 μm, and further is preferably 2 μm to 8 μm.

The method for making the honeycomb membrane is not limited. Examples of methods for making honeycomb membranes include: ｃA production method of forming through-holes by growing water droplets in ｃA coating film comprising ｃA polymer and ｃA solvent, disclosed in japanese patent nos. 4,734,157, 4,945,281, 5,405,374 and 5,422,230 and japanese patent application laid-open (JP- ｃA) No. 2011-74140; and a method for forming a honeycomb film by forming through-holes by performing etching treatment or punching treatment on a film made of a resin.

Examples of the material of the honeycomb film 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 (polyacylamine), 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), poly (glycerol sebacate), and polyacrylamide (polyacrylamine). 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 honeycomb membrane is preferably polybutadiene, polyurethane, polycarbonate, or polylactic acid from the viewpoint of self-supporting property, and is preferably polylactic acid, a polylactic acid-polyglycolic acid copolymer, or a polylactic acid-polycaprolactone copolymer from the viewpoint of transplantation and maintenance of the cell layer.

From the viewpoint of cell adhesion properties, each surface of the honeycomb membrane is preferably coated with at least kinds 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, nephronexin, basement membrane matrix, recombinant peptide, and polylysine, and at least the region where the cell layer is disposed is coated.

[ th cell and second cell ]

The cell and the second cell are, for example, two cells selected from the group consisting of a parenchymal cell (e.g., a hepatic parenchymal cell or a pancreatic parenchymal cell), a stromal cell (e.g., a pericyte), a muscle cell (e.g., a smooth muscle cell, a cardiac muscle cell or a skeletal muscle cell), a fibroblast, a nerve cell, a glial cell, an endothelial cell (e.g., a vascular endothelial cell or a lymphatic endothelial cell), an epithelial cell (e.g., an alveolar epithelial cell, an oral epithelial cell, a biliary tract epithelial cell, an intestinal epithelial cell, a pancreatic duct epithelial cell, a renal tubular epithelial cell or a placental epithelial cell), and a stem cell (e.g., a mesenchymal stem cell).

In the cell stack according to the present invention, or more kinds of cells (referred to as third cells) other than th cells and second cells may be contained in cell layers or two cell layers in the cell stack according to the present invention, for example, th cells are parenchymal cells, second cells are stromal cells, and third cells are nerve cells, which may be contained in or two cell layers.

Even if cell layers containing th cells contain the same second cells as those contained in the other cell layers, the structure is still within the present invention as long as the cells contained in the cell layers and the cells contained in the other cell layers can be distinguished, for example, according to the content ratio between the two cells, for example, the present invention includes a configuration in which the cells contained in the cell layers are parenchymal cells and stromal cells (content ratio is 9:1), and the cells contained in the other cell layers are parenchymal cells and stromal cells (content ratio is 1: 9).

The cell laminate according to the present invention is a tissue model imitating a tissue in a living body and is included in the living tissue model device according to the present invention therefore, th cells and second cells, and if necessary, third cells are selected according to the tissue in a living body to be imitated in animal tissue, a basement membrane is generally present between a cell layer and another cell layer in the cell laminate (used as a tissue model) according to the present invention, a honeycomb membrane corresponds to a basement membrane.

An example of a tissue model simulating a tissue in a living body is a blood vessel wall model the blood vessel wall model according to the present invention includes a honeycomb membrane, a vascular endothelial cell layer disposed on a surface of the honeycomb membrane, and a smooth muscle cell layer or a mesenchymal stem cell layer disposed on another surface of the honeycomb membrane.

The vascular wall model according to the present invention is preferably configured such that the FITC-dextran 70 permeability from the vascular endothelial cell layer side to the smooth muscle cell layer side or the mesenchymal stem cell layer side is 0% to 10% of the FITC-dextran 70 permeability of the honeycomb membrane itself, more preferably 0% to 5% of the FITC-dextran 70 permeability of the honeycomb membrane itself, and further is preferably 0% to 2% of the FITC-dextran 70 permeability of the honeycomb membrane itself.

Hereinafter, a method for analyzing permeability of FITC-dextran 70 in a blood vessel wall model will be described.

Another example of a tissue model that mimics tissue in an organism is a pathology replication model in which cells with genetic mutations or cells from a patient are used as at least of the th cell or the second cell.

The living tissue model device comprising the above cell laminate can be used as a device for drug evaluation or pathology evaluation, or can replace a test device for animal experiments. Next, a method of evaluating a test substance using a living tissue phantom apparatus will be described as an application of the living tissue phantom apparatus according to the present invention.

< method of evaluating test substance >

The biopsy model device according to the present invention can be used as a means for evaluating the influence of a test substance on a cell tissue. Specifically, with the living tissue model device according to the present invention, the influence of the test substance on the cellular tissue is evaluated by the following steps:

a step of adding a test substance to at least of the th liquid compartment or the second liquid compartment, and

For example, the test substance is evaluated according to the following modes (a) and (b).

(a) Mode for quantifying chemical substance secreted from cells of cell stack

The cells in the -side cell layer facing the liquid compartment to which the test substance has been added secrete a chemical substance (including leakage of intracellular components due to cell breakage) in response to the test substance, as a result, the liquid compartment to which the test substance has been added contains a substance secreted by the cells, and the cells in the -side cell layer opposite to the cell layer facing the liquid compartment to which the test substance has been added secrete the chemical substance due to any of intercellular interaction (i.e., signal transduction due to lytic factors) between the cell layer on the surface and the cell layer on the other surface or intercellular contact between the cell layer on the surface and the cell layer on the other surface.

(b) Mode for quantifying chemical substance or cell leaked from side of cell laminate to side of cell laminate

The cells in the cell layer on the side opposite the liquid compartment to which the test substance has been added change their morphology in response to the test substance (including cell damage) and may create cracks in the cell layer, as a result of which the chemical substance or cells contained in the liquid composition stored in the liquid compartment to which the test substance has been added leak to another liquid compartment, the chemical substance or cells that have leaked to another liquid compartment are quantified, and the obtained amount is used to determine whether or not the test substance has an effect on the cell tissue and the degree of effect.

In the mode (b), after a test substance is added to a liquid compartment, a tracer is added to the liquid compartment to which the test substance has been added, and the amount of the tracer leaked to another liquid compartment is quantified.A test substance is added to a liquid compartment, and then cultured, for example, at 37 ℃ for 30 minutes to 24 hours, and a tracer is added.

In this mode (a) and (b), the living tissue phantom apparatus according to the present invention is superior to the conventional living tissue phantom apparatus in the following respects.

In addition, in the cell laminate having cell layers on both sides of the TE membrane, even if the morphology of the cells in the cell layer changes to generate cracks in the cell layer, the possibility that the pores on the TE membrane closed by the cell layer are penetrated is low, even if the barrier function of the cell layer disappears due to the test substance, the barrier function itself of the TE membrane functions and leakage of the test agent can be prevented.

The living tissue model device according to the present invention includes a cell laminate including cell layers on both sides of a honeycomb membrane having a high opening ratio, wherein the cell laminate includes cell layers on both sides of the honeycomb membrane, the cell layer on the surface and the cell layer on the other surface are relatively active, and it is presumed that the active cell-cell interaction causes a cell layer on the side opposite to the side facing a liquid cell to which a test substance has been added to show a desired reaction and secretes a desired chemical substance, and further, in the cell laminate including cell layers on both sides of the honeycomb membrane, when the morphology of cells in the cell layer changes to generate cracks in the cell layer, the possibility that the pores on the honeycomb membrane closed by the cell layer are penetrated is high, and therefore, the barrier function of denier cell layer is lost by the test substance, and the possibility that a tracer leaks is detected, therefore, with the living tissue model device according to the present invention, the influence of the substance on the cell tissue can be evaluated with high sensitivity.

The vascular wall modeling apparatus according to the present invention is useful as a means for evaluating the influence of a test substance on the vascular wall. Specifically, with the vascular wall model device according to the present invention, the influence of the test substance on the vascular wall is evaluated by the following steps:

adding a test substance to at least of the th or second liquid compartment, and

(i) at least of the steps of (i) quantifying at least of the chemical substances contained in the th liquid compartment or the cells contained in the th liquid compartment, and (ii) quantifying at least of the chemical substances contained in the second liquid compartment or the cells contained in the second liquid compartment, the test substance is evaluated, for example, according to the following mode (a-1) or (b-1).

(a-1) mode for quantifying chemical substance secreted from cell in vascular wall model

This mode is performed in the same manner as the above-described mode (a).

(b-1) quantitative mode of chemical substance or cell leaking from the side of the blood vessel wall model device to the other side of the blood vessel wall model device

This mode is performed in the same manner as the above-described mode (b). example is the above-described mode using a tracer.

In the blood vessel wall model device of example as mode (b-1), blood, a liquid composition containing red blood cells, or a liquid composition containing at least selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres and mimicking the blood was used and stored in at least of the th liquid compartment or the second liquid compartment, in this mode, S adds a test substance to the liquid composition containing blood, a liquid composition containing red blood cells, or a liquid composition containing at least selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres and quantifies at least of the amount of red blood cells leaked to another liquid compartment, the amount of hemoglobin leaked to another liquid compartment, or the amount of at least selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres leaked to another liquid compartment.

In example of the above mode, using a blood vessel wall model in which blood, a liquid composition containing erythrocytes, or a liquid composition containing at least kinds of substances selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres and mimicking blood is stored in a liquid compartment located on the side facing the vascular endothelial cell layer, a test substance is added to the liquid compartment located on the side facing the vascular endothelial cell layer, and the amount of erythrocytes leaking to the liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, the amount of hemoglobin leaking to the liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, or the amount of at least kinds of substances selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres, leaking to the liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, is quantitatively evaluated.

When a substance to be detected has an effect on vascular endothelial cells, the vascular endothelial cells react to the substance to be detected (including damage of vascular endothelial cells), and the permeability of the chemical substance to the vascular wall is improved, as a result, at least kinds of red blood cells or at least kinds of red blood cells selected from the group consisting of dextran, evans blue, fluorescein sodium salt, and FITC-microspheres, contained in a liquid composition located on the side facing the vascular endothelial cell layer leak into a liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, when the substance to be detected also has hemolytic toxicity, hemoglobin flows out from the red blood cells, and the hemoglobin leaks into a liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, the amount of red blood cells leaking into a liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, the amount of hemoglobin leaking into a liquid compartment located on the side facing the smooth muscle cell layer or the mesenchymal stem cell layer, and whether the amount of the liquid composition leaking out of hemoglobin contained in the liquid compartment and the liquid composition is selected from the group consisting of hemoglobin , the liquid compartment located on the opposed side, the hemoglobin layer, and the FITC-enriched liquid compartment.

The vessel wall model device according to the invention can be used as a means of evaluating the barrier function of a vessel wall model. For example, using a cell culture insert-type device in which the filter portion is a blood vessel wall model, the barrier function of the blood vessel wall model is evaluated by analyzing FITC-dextran 70 permeability in the following manner

FITC-dextran 70 was added to the inside of the cell culture insert and cultured at 37 ℃, and the amount of FITC-dextran 70 that leaked to the outside of the cell culture insert within 10 minutes (i.e., the fluorescence intensity of FITC outside the cell culture insert was measured) was measured using a cell culture insert-type device in which the filtration section was the honeycomb membrane itself, and the amount of FITC-dextran 70 that leaked to the outside of the cell culture insert was measured in the same procedure as described above (i.e., the fluorescence intensity of FITC outside the cell culture insert was measured), the ratio of the fluorescence intensity obtained in the first measurements to the fluorescence intensity obtained in the latter measurements was measured, i.e., the ratio of the fluorescence intensity obtained in the latter measurements was regarded as% and the RFI intensity was preferably expressed as a smaller ratio, i.e., the RFI intensity is expressed as 0% to 10%, and the RFI barrier function is preferably expressed as 0% to 539 2%, which is expressed as a smaller function of RFI 0% to 5%, preferably expressed as a smaller function of RFI 0% RFI.

< method for producing cell laminate and living tissue model device >

The living tissue phantom device according to the present invention is produced, for example, by a method comprising a step of mounting a cell laminate as a spacer in the living tissue phantom device and a step of obtaining a cell laminate by culturing different types of cells on each surface of a honeycomb membrane, or a method comprising a step of constructing a spacer portion in the living tissue phantom device into a honeycomb membrane and a step of forming a cell laminate by culturing different types of cells on each surface of the honeycomb membrane.

In the manufacturing method according to the present invention, a container having a bottom portion and a side wall portion standing from an edge of the bottom portion, a honeycomb film, and a holding member configured to hold the honeycomb film so that the honeycomb film faces an inner bottom surface of the container and is held at a position not in contact with the inner bottom surface are used. Hereinafter, the vessel is referred to as "culture vessel".

The production method according to the present invention includes culturing cells on both surfaces of a honeycomb membrane using a culture vessel, the honeycomb membrane, and a holding member, thereby producing a cell laminate having cell layers on both surfaces of the honeycomb membrane.

First, a culture vessel, a honeycomb film and a holding member used in the production method according to the present invention will be described. The following examples of the culture vessel, the honeycomb membrane and the holding member correspond to preferred examples of the cell culture insert-type apparatus.

The culture vessel is, for example, a tray, a multi-tray, or a multi-well tray. The bottom of the culture vessel has, for example, a circular, rectangular or square shape. The material of the culture vessel is, for example, polystyrene, polycarbonate, polyester or glass. The culture vessel preferably has high transparency.

The inner bottom surface of the culture vessel is preferably flat. The inner bottom surface of the culture vessel preferably has properties that do not allow cells to adhere to the inner bottom surface. Therefore, the inner bottom surface of the culture vessel is preferably not subjected to corona discharge treatment or protein coating treatment. In order to reduce cell adhesion, the inner bottom surface of the culture vessel may be coated with a polymer of phosphorylcholine group or polyethylene glycol, for example. Similarly to the inner bottom surface, the inner side surface of the culture container is also preferably provided with a property that cells do not adhere to the inner side surface.

The honeycomb membrane used in the production method according to the present invention has the same meaning as that of the honeycomb membrane included in the cell laminate, and preferred examples thereof are also the same. In the manufacturing method according to the present invention, the honeycomb membrane is a scaffold to which cells adhere and proliferate.

The higher opening ratio of the honeycomb membrane and the thinner thickness of the honeycomb membrane provide at least of more active intercellular interactions (i.e., signaling by lytic factors) between cells on the surface and the further surface or more active intercellular contacts between cells on the surface and the further surface, respectively.

The holding member is configured to hold the honeycomb film so that the honeycomb film faces the inner bottom surface of the container and is held at a position not in contact with the inner bottom surface.

As the material of the holding member, for example, polycarbonate, polystyrene, and polyester are preferable from the viewpoints of high transparency, chemical stability in a liquid medium, and light weight.

The shape of the holding member is not limited. The holding member includes, for example, a portion configured to hold the honeycomb membrane and a portion configured to contact the culture container. The holding member is, for example, a linear member having a protruding portion to be engaged with an edge of the side wall portion of the culture container, a rod-like member, or a hollow cylindrical member.

As for the form of the holding member, the holding member is, for example, a member including:

a hollow cylindrical part configured to hold the porous membrane at an axial end of the hollow cylindrical part (the cylindrical part having an outer diameter smaller than the inner diameter of the culture container and the axial length of the hollow cylindrical part being shorter than the height of the side wall part of the culture container), and

a projection projecting radially outward from the other axial end of the hollow cylindrical part (the projection is configured to abut against the edge of the side wall part of the culture container), and the embodiments will be described below with reference to the drawings.

Fig. 4A shows example of the holding member 40 as the holding member in a state of being combined with the honeycomb film 20( example of the honeycomb film), fig. 4A is a perspective view of the holding member 40, and fig. 4B is a perspective view showing a state in which the holding member 40 combined with the honeycomb film 20 is mounted in the culture container 60( example of the culture container).

The holding member 40 includes a hollow cylindrical portion 42 and a protruding portion 44, the honeycomb film 20 is disposed at an axial end portion of the hollow cylindrical portion 42, the honeycomb film 20 has a size that closes at least an opening located at an end of the hollow cylindrical portion 42, the honeycomb film 20 is adhered to an end of the hollow cylindrical portion 42 by thermal compression bonding, ultrasonic welding, laser welding, an adhesive, or a double-sided tape, or the honeycomb film 20 may be fixed to an end of the hollow cylindrical portion 42 by an annular fixing member that is adhered to an outer side surface of the hollow cylindrical portion 42.

The hollow cylindrical portion 42 has an outer diameter smaller than the inner diameter of the culture container 60, and can be inserted into the culture container 60 (i.e., a space defined by the bottom portion 62 and the side wall portion 64). The axial length of the hollow cylindrical part 42 is shorter than the height of the side wall part 64 of the culture container 60. Therefore, the honeycomb membrane 20 does not contact the bottom 62 of the culture vessel 60.

The hollow cylindrical portion 42 has a wall continuous in the circumferential direction and the axial direction. This structure enables the liquid to be stored in the space defined by the honeycomb film 20 and the hollow cylindrical portion 42. However, a slit may be provided in the wall of the hollow cylindrical portion 42 at a position close to the protruding portion 44. The shape of the inner surface of the hollow cylindrical portion 42 is, for example, a cylindrical shape, a polygonal columnar shape, a truncated cone shape, or a truncated pyramid shape.

The protruding portion 44 protrudes outward in the radial direction of the hollow cylindrical portion 42 at the axial end portion of the hollow cylindrical portion 42 opposite to the end portion provided with the honeycomb film 20. For example, the three protrusions 44 are provided at intervals of about 120 ° along the circumferential direction of the hollow cylindrical portion 42. However, the number and shape of the projections 44 are not limited thereto. The protruding portion 44 may have a ring shape continuous in the axial direction of the hollow cylindrical portion 42.

The projection 44 has a projection length that allows the projection 44 to engage with the edge of the side wall portion 64 of the culture container 60 when the holding member 40 is inserted into the inside of the culture container 60. The holding member 40 is fixed to the edge of the side wall portion 64 of the culture vessel 60 by the projection 44.

In general, a culture device having the shape shown in FIG. 4A is referred to as a cell culture insert.

The following describes a production method according to the present invention. In the present invention, the scope of the "step" includes an independent step and a target step that can achieve a desired object even though it cannot be clearly distinguished from other steps.

The production method according to the present invention uses a culture container, a honeycomb film, and a holding member, and the production steps include the following steps (a) and (B). fig. 5 is a schematic diagram of example showing the production method according to the present invention, and is a schematic diagram for explaining the steps (a) and (B). in fig. 5, arrow G indicates the direction of gravity.

And a step (A) of culturing th cells 11 in a liquid culture medium in contact with the inner bottom surface of the culture container 6 and the surface of the honeycomb membrane 2, in a state in which the honeycomb membrane 2 is held by the holding member 4 at a position not in contact with the inner bottom surface of the culture container 6 so as to face the inner bottom surface, and the bottom of the culture container 6 is positioned on the upper side and the honeycomb membrane 2 is positioned on the lower side in the direction of gravity G.

And (B) culturing th cells 12 on the upper surface of the honeycomb membrane 2 and culturing second cells 11 on the lower surface of the honeycomb membrane 2 while the honeycomb membrane 2 is held by the holding member 4 at a position not in contact with the inner bottom surface of the culture container 6 so as to face the inner bottom surface and the bottom of the culture container 6 is positioned below the honeycomb membrane 2 in the direction of gravity G.

"culturing" in the present invention does not necessarily involve cell proliferation, and maintaining cells in a living state, whether proliferation exists or not, is included in the scope of the term.

In the state employed in the step (A), the honeycomb film 2 is located on the lower side and the bottom of the culture container 6 is located on the upper side in the direction of gravity G, and in the step (A), the cell suspension including the th cell 11 is disposed between the culture container 6 and the honeycomb film 2, the cell suspension is brought into contact with the inner bottom surface of the culture container 6 and the surface of the honeycomb film 2, and the th cell 11 is cultured in this state, since surface tension acts between the inner bottom surface of the culture container 6 and the liquid culture medium contained in the cell suspension, the liquid culture medium remains in the honeycomb film 2, and dripping of the liquid culture medium through the hole of the honeycomb film 2 is reduced, and therefore, the honeycomb film having a high aperture ratio can be used to produce a cell laminate, and the honeycomb film 2 is preferably held by the holding member 4 in a position close to the inner bottom surface of the culture container 6, in a state where the honeycomb film 2 is opposed to and oriented parallel or substantially parallel to the inner bottom surface of the culture container 6, and the distance between the honeycomb film 2 and the inner bottom surface of the culture container 6 is 0.5mm to 10mm, for example.

In the step (A), the th cells 11 in the liquid medium migrate in the direction of gravity G due to their own weight and adhere to the honeycomb membrane 2, and the step (A) is a step of adhering-culturing the th cells 11 to the honeycomb membrane 2.

Cell culture conditions in step (A)May be conventional cell culture conditions. For example, CO at a temperature of 37 ℃ and 5% (v/v) can be used₂Concentration incubator (for example, CO manufactured by Panasonic Corporation)₂An incubator) during the culture, it is preferable that the period until the th cell 11 becomes stable in adhesion to the honeycomb membrane 2.

In the state employed in the step (B), the honeycomb membrane 2 is located on the upper side and the bottom of the culture container 6 is located on the lower side in the direction of gravity G, in the step (B), the th cell 11 is cultured on the lower surface of the honeycomb membrane 2, and the th cell 11 to be cultured on the lower surface of the honeycomb membrane 2 is the th cell 11 to be cultured on the surface of the honeycomb membrane 2 located on the side facing the inner bottom surface of the culture container 6, and the cell is continued to be cultured in the step (B).

The cell culture conditions in the step (B) may be conventional cell culture conditions. For example, CO at a temperature of 37 ℃ and 5% (v/v) can be used₂Culturing in a concentration incubator. The period of culture is preferably until the cells reach confluency (confluency) on both sides of the honeycomb membrane 2. The cell confluence can be detected, for example, by optical microscopy. During the cultivation, the medium may be changed to another medium.

examples of a production method including steps (A) and (B) will be described with reference to FIG. 6, an exemplary method shown in FIG. 6 is a production method using a culture apparatus having a shape shown in FIG. 4B, headings (1) to (5) in FIG. 6 correspond to steps (1) to (5), respectively, in FIG. 6, an arrow G indicates a direction of gravity, according to an exemplary method including steps (1) to (5), the production method including steps (A) and (B) can be easily realized, and an exemplary method including steps (1) to (5) is a method of producing a cell laminate and at the same time, a method of producing a example cell culture insert type apparatus as a living tissue model apparatus.

Step (1) of supplying a cell suspension containing th cells 11 to the inner bottom surface of the culture container 6.

In the step (1), it is preferable that the cell suspension is supplied to the inner bottom surface so as not to contact the inner side surface of the culture vessel 6 because it is necessary to prevent the cell suspension from falling down along the inner side wall of the culture vessel 6 in the step (3). Another method for preventing the cell suspension from falling down along the inner side wall of the culture vessel 6 is, for example, to set the dimension of the honeycomb membrane 2 on the main surface thereof to a dimension of contacting the inner side surface of the culture vessel 6 over the entire circumference.

In the step (1), the amount of the cell suspension supplied to the inner bottom surface of the culture vessel 6 is preferably the same as the volume of the space sandwiched between the inner bottom surface of the culture vessel 6 and the honeycomb membrane 2, and the seeding density of the th cell 11 is, for example, 1.0X 10 based on the area of the honeycomb membrane 2³～1.0×10⁶Cells/cm²。

Step (2): the holding member 4 having the honeycomb membrane 2 is disposed in the culture container 6, and the honeycomb membrane 2 is brought into contact with the cell suspension supplied to the inner bottom surface of the culture container 6.

As a result of the execution of the step (2), the cell suspension including the th cells 11 was brought into contact with the inner bottom surface of the culture vessel 6 and the surface of the honeycomb membrane 2 (in other words, the cell suspension was sandwiched between the inner bottom surface of the culture vessel 6 and the surface of the honeycomb membrane 2). in the following explanation of the production method, the apparatus including the holding member 4 of the honeycomb membrane 2 and the culture vessel 6 as bodies was referred to as "culture apparatus".

In the step (3), the th cells 11 are cultured between the inner bottom surface of the culture container 6 and the honeycomb membrane 2 in a state where the bottom of the culture container 6 is located on the upper side and the honeycomb membrane 2 is located on the lower side in the direction of gravity G.

The culture apparatus is inverted in a state where the holding member 4 provided with the honeycomb membrane 2 is attached to the culture container 6, and then the culture apparatus is left standing in the incubator, whereby the step (3) is achieved, the th cells 11 contained in the cell suspension migrate in the direction of gravity G by their own weight and adhere to the honeycomb membrane 2.

Step (4): the second cells 12 are cultured on the upper surface of the honeycomb membrane 2 in a state where the bottom of the culture container 6 is positioned on the lower side and the honeycomb membrane 2 is positioned on the upper side in the direction of gravity G.

The culture apparatus is taken out of the incubator and is inverted again, and thereafter a cell suspension containing the second cells 12 is seeded on the honeycomb membrane 2, thereby realizing the process (4). Second cellThe seeding density of 12 is, for example, 1.0X 10³～1.0×10⁶Cells/cm²Liquid medium is preferably added to the th cell 11 side before or after seeding the second cell 12.

In the step (5), the th cell 11 is cultured on the lower surface of the honeycomb membrane 2 and the second cell 12 is cultured on the upper surface of the honeycomb membrane 2 in a state where the bottom of the culture container 6 is positioned on the lower side and the honeycomb membrane 2 is positioned on the upper side in the direction of gravity G.

The step (5) is achieved by leaving the culture apparatus standing in the incubator following the step (4). during the step (5), the medium may be replaced with another medium.when at least of the th cells 11 or the second cells 12 are stem cells, a differentiation-inducing factor that induces differentiation into desired somatic cells is added to the medium.

Through the steps (1) to (5), a cell laminate including the honeycomb membrane 2, a cell layer including the th cell 11 and disposed on the surface of the honeycomb membrane 2, and a cell layer including the second cell 12 and disposed on the other surface of the honeycomb membrane 2 are obtained.

Hereinafter, a cell used in the production method according to the present invention will be described.

and second cells are, for example, two cells selected from the group consisting of parenchymal cells (e.g., liver parenchymal cells or pancreas parenchymal cells), stromal cells (e.g., pericytes), muscle cells (e.g., smooth muscle cells, cardiac muscle cells, or skeletal muscle cells), fibroblasts, nerve cells, glial cells, endothelial cells (e.g., vascular endothelial cells or lymphatic endothelial cells), and epithelial cells (e.g., alveolar epithelial cells, oral epithelial cells, bile duct epithelial cells, intestinal epithelial cells, pancreatic duct epithelial cells, kidney epithelial cells, renal tubular epithelial cells, or placental epithelial cells), and cells capable of differentiating into these cells (e.g., progenitor cells, mesenchymal stem cells, or pluripotent stem cells).

Examples of pluripotent stem cells that can be used as the th cell or the second cell include Embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, Embryonic Germ (EG) cells, Embryonic Carcinoma (EC) cells, Multipotent Adult Progenitor (MAP) cells, Adult Pluripotent Stem (APS) cells, and multi-lineage differentiation stress-tolerant (Muse) cells.

In the manufacturing method according to the present invention, cells (also referred to as "third cells", which may be or more) different from the th cell and the second cell may be co-cultured with at least of the th cell or the second cell, and as a result of the co-culture, a cell layer including the third cell and the th or second cell is formed on the side or both sides of the honeycomb membrane, and in an exemplary combination, the th cell is a parenchymal cell, the second cell is a stromal cell, and the third cell is a neural cell.

In a method of manufacturing a model tissue for simulating a living body tissue according to the present invention, a combination of th cells and second cells, and if necessary, a third cell, may be selected according to the living body tissue to be simulated, in example of the method of manufacturing the present invention, th cells are smooth muscle cells or cells differentiated into smooth muscle cells, and second cells are vascular endothelial cells or cells differentiated into vascular endothelial cells, in another example of the method of manufacturing the present invention, th cells are mesenchymal stem cells, and second cells are vascular endothelial cells or cells differentiated into vascular endothelial cells, the method of manufacturing the present invention provides kinds of cell laminates in which an intravascular endothelial cell layer is disposed on a surface of a honeycomb membrane, and a smooth muscle cell layer or an mesenchymal stem cell layer is disposed on another surface of the honeycomb membrane, that is, a vascular wall model.

For the purpose of reproducing the pathology, cells having a gene mutation or cells from the patient were used as at least of the th cell or the second cell.

Examples of the specific medium include media that are optimized for the cell type by adding cell growth factors to a basal medium for mammalian cells, such as Dulbecco's Modified Eagle's Medium (DMEM), dalberg modified Eagle's medium: nutrient mixture F-12 (DMEM: F-12), Eagle's basic ingredient medium (EMEM), basic ingredient medium α (α), or Eagle's basic ingredient medium (BME), which are commercially available.

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Biopsy phantom device, blood vessel wall phantom device, and method for evaluating test substance

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