JP2021176287A - Cell structure and method for producing the same - Google Patents

Abstract

To provide a cell structure that has a smooth muscle cell, a vascular endothelial cell and a cell matrix, and includes an immunocyte, and a method for producing the structure.SOLUTION: A method for producing a cell structure includes an immunocyte includes: a manufacturing process of a cell structure which includes mixing a smooth muscle cell and a fragmented extracellular matrix in aqueous medium and incubating them to form a smooth muscle cell layer, and bringing a vascular endothelial cell into contact with the smooth muscle cell layer and incubating them to form a vascular endothelial cell layer contacting the smooth muscle cell layer; a process of bringing an inflammation promoter into contact with the cell structure; and a process of bringing an immunocyte into contact with the cell structure.SELECTED DRAWING: None

Description

The present invention relates to a cell structure and a method for producing the same.

Arteriosclerosis is a problem that people should avoid in order to live in good health. In addition, arteriosclerosis is said to be related to the infiltration of immune cells that are attracted to the inflammatory reaction that occurs inside the blood vessel wall and continue to stay there.

Research is being conducted to construct a vascular model containing vascular smooth muscle cells that can be used for the study of vascular diseases such as arteriosclerosis and the screening of drugs for prevention or treatment. For example, Non-Patent Document 1 discloses that a three-dimensional cell structure composed of a smooth muscle cell layer and an endothelial cell layer is produced by an alternating stacking method (Layer-by-layer: Lb method).

There is still a need for a cell structure that can contain more extracellular matrix and that can be used as a vascular disease model by including immune cells and a method for producing the same.

M. Matsusaki, U. Yokoyama et al., Atherosclerosis, 2014, 233, 590.

An object of the present invention is to provide a cell structure having smooth muscle cells, vascular endothelial cells and an extracellular matrix and containing immune cells, and a method for producing the same.

That is, the present invention relates to, for example, the following inventions.

[1] A method for producing a cell structure containing immune cells.
Smooth muscle cells and fragmented extracellular matrix are mixed and incubated in an aqueous medium to form a smooth muscle cell layer, and vascular endothelial cells are contacted and incubated with the smooth muscle cell layer to incubate and smooth. Forming a vascular endothelial cell layer in contact with a muscle cell layer, and a process for producing a cell structure containing
The step of bringing the pro-inflammatory agent into contact with the cell structure and
A method comprising contacting an immune cell with the cell structure.
[2] The production method according to [1], wherein the step of contacting the immune cells with the cell structure comprises contacting the immune cells with the vascular endothelial cell layer.
[3] The production method according to [1] or [2], wherein the fragmented extracellular matrix has the sequence represented by SEQ ID NO: 1 (GVGVP sequence) and / or the sequence represented by SEQ ID NO: 2 (VGVAPG sequence). ..
[4] The production method according to [3], wherein the fragmented extracellular matrix is elastin.
[5] The production method according to any one of [1] to [4], wherein the pro-inflammatory agent is Angiotensin II or lipoprotein.
[6] The production method according to [5], wherein the lipoprotein is a denatured lipoprotein.
[7] A smooth muscle cell layer containing smooth muscle cells and a fragmented extracellular matrix,
Has a vascular endothelial cell layer, which contains vascular endothelial cells,
The smooth muscle cell layer and the vascular endothelial cell layer are in contact with each other.
A cell structure that contains immune cells.
[8] The cell structure according to [7], wherein the smooth muscle cell layer or the vascular endothelial cell layer contains immune cells.
[9] The cell structure according to [7] or [8], wherein the immune cell contains a lipoprotein inside.
[10] A method for evaluating the effect of a drug using the cell structure according to [7] or [8].
[11] An administration step of administering the drug to the cell structure and
The evaluation method according to [10], which includes an evaluation step of evaluating the effect of the drug based on changes in the cell structure to which the drug has been administered.
[12] The evaluation method according to [10] or [11], wherein the agent is an arteriosclerosis inhibitor or an arteriosclerosis promoter.
[13] In a cell structure having a smooth muscle cell layer containing smooth muscle cells and a fragmented extracellular matrix, and a vascular endothelial cell layer containing vascular endothelial cells.
The process of contacting the pro-inflammatory agent,
The process of contacting immune cells,
A method for evaluating the effect of a drug, which comprises an administration step of administering the drug to a cell structure and an evaluation step of evaluating the effect of the drug based on changes in the cell structure to which the drug is administered.
[14] The evaluation method according to [13], wherein the agent is an arteriosclerosis inhibitor or an arteriosclerosis promoter.
[15] The evaluation method according to [13] or [14], wherein the step of contacting the immune cells with the cell structure comprises contacting the immune cells with the vascular endothelial cell layer.

According to the present invention, it is possible to provide a cell structure having smooth muscle cells, vascular endothelial cells and a cell matrix and containing immune cells, and a method for producing the same. Since the cell structure contains immune cells, it can be used as a vascular wall model with vascular disease. Further, the content of the extracellular matrix contained in the cell structure can be easily adjusted, and it is possible to produce a cell structure having a high extracellular matrix content.

It is a photograph which shows the observation result of the cell structure by CD11b staining and CD14 staining in Test Example 3. It is a graph which compared the fluorescence area in the cell structure by CD11b staining and CD14 staining in Test Example 3. It is a photograph which shows the observation result (observation from above) of the cell structure by CD31 staining, CD54 staining and CD106 staining in Test Example 4. It is a graph which compared the fluorescence area by CD54 staining in Test Example 4. In Test Example 4, (a) a graph comparing the fluorescence area by CD54 staining and (b) a graph comparing the ratio of the fluorescence area when the fluorescence area is 1.0 when Angiotensin II is not included. Is. It is a photograph showing the observation result of the cell structure by hematoxylin and eosin (HE) staining in Test Example 5, and the photograph which partially enlarged the part including the endothelial cell layer. It is a graph which shows the change of the thickness of the endothelial cell layer of the cell structure with respect to the concentration of acetylated low density lipoprotein in Test Example 5. It is a photograph which shows the observation result of the cell structure by (a) CD31 staining and (b) the observation result of the cell structure by α-SMA staining in Test Example 5. It is a photograph which shows the observation result of the cell structure by CD31 staining in Test Example 6. 6 is a graph showing the change in the thickness of the endothelial cell layer of the cell structure with respect to the concentration of high-density lipoprotein (HDL) in Test Example 6.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Cell structure]
The cell structure according to the present embodiment has a smooth muscle cell layer containing smooth muscle cells and a fragmented extracellular matrix, and a vascular endothelial cell layer containing vascular endothelial cells, and includes immune cells. The smooth muscle cell layer and the vascular endothelial cell layer are in contact with each other. The cell structure according to the present embodiment is a biological tissue model having a function similar to the function of at least a part of the blood vessel wall having a vascular disease and / or a structure similar to the structure of at least a part of the blood vessel wall having a vascular disease. It can be used as a vascular wall tissue having a vascular disease.

As used herein, the term "cell structure" means an aggregate of cells in which cells are three-dimensionally arranged (massive cell population), which is artificially produced by cell culture. Extracellular matrix components may be arranged between at least some cells. In the cell structure, there may be a portion where cells are in direct contact with each other.

The shape of the cell structure is not particularly limited, and is, for example, sheet-like, spherical, substantially spherical, ellipsoidal, approximately ellipsoidal, hemispherical, approximately hemispherical, semicircular, approximately semicircular, rectangular parallelepiped. , Approximately rectangular parallelepiped, etc. Here, the biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and is more complicated in composition than the cell structure. Therefore, the cell structure and the living tissue can be easily distinguished. Further, the cell structure may be assembled in a mass in a state of being adhered to the support, or may be aggregated in a mass in a state of not being adhered to the support.

The cells may be somatic cells or germ cells. Further, the cell may be a stem cell, or may be a cultured cell such as a primary cultured cell, a subcultured cell, and a cell line cell. As used herein, the term "stem cell" means a cell having self-renewal ability and pluripotency. Stem cells include pluripotent stem cells capable of differentiating into arbitrary cell tumors and tissue stem cells (also called somatic stem cells) capable of differentiating into specific cell tumors. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include mesenchymal stem cells (eg, bone marrow-derived stem cells), hematopoietic stem cells, and neural stem cells.

The total number of cells constituting the cell structure according to the present embodiment is not particularly limited, and is appropriately considered in consideration of the thickness and shape of the cell structure to be constructed, the size of the cell culture vessel used for construction, and the like. It is determined.

(Smooth muscle cell layer)
The smooth muscle cell layer in the cell structure according to the present embodiment contains smooth muscle cells and a fragmented extracellular matrix.

Smooth muscle cells are spindle-shaped cells that make up smooth muscle, and are mainly distributed in the digestive tract, bladder, uterus, and blood vessel wall. Smooth muscle cells include, for example, aortic smooth muscle cells (Aorta-SMC), coronary arterial smooth muscle cells (CASMC), umbilical artery smooth muscle cells (UASMC), pulmonary arterial smooth muscle cells (PASMC), tracheal smooth muscle cells (TSMC). , Bronchial smooth muscle cells (BSMC), uterine smooth muscle cells (HUtSMC). The smooth muscle cells constituting the cell structure may be primary cells (primary smooth muscle cells) collected from the aorta of an animal (for example, human), cells obtained by culturing primary cells, or primary cells. It may be a cultured cell line in which cells are established, or may be cells artificially differentiated from stem cells. Examples of the cultured cell line include a cell line of human umbilical artery-derived smooth muscle cells (UASMC) manufactured by Lonza. Examples of stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells and the like. The smooth muscle cells contained in the cell structure according to the present embodiment may be non-cancerous.

The ratio (X ₁ / X ₀ x 100) of the number of smooth muscle cells (X ₁ ) to the total number of cells (X ₀ ) in the cell structure is 5% or more, 10% or more, 15% or more, 20% or more. , 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, 95% or less, 90% or less, It may be 80% or less or 75% or less. The ratio (X ₁ / X ₀ × 100) of the number of smooth muscle cells (X ₁ ) to the total number of cells (X ₀ ) in the cell structure is 60% or more from the viewpoint of being more suitable as a vascular wall tissue. It may be 80% or less, or 60% or more and 70% or less.

The thickness of the smooth muscle cell layer is preferably 20 μm to 500 μm, more preferably 50 μm to 200 μm. The upper limit of the thickness of the smooth muscle cell layer is not particularly limited, but may be, for example, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less. May be good. From the viewpoint of using as a blood vessel wall model having vascular disease (for example, arteriosclerosis), the thickness of the smooth muscle cell layer is preferably 50 μm to 150 μm.

Here, the "thickness of the cell layer" means a distance in a direction perpendicular to the main surface when the cell structure is in the form of a sheet or a rectangular parallelepiped.

When the cell structure is spherical or substantially spherical, it means the distance in the radial direction thereof. Furthermore, when the cell structure is ellipsoidal or substantially ellipsoidal, it means the distance in the minor axis direction.

(Fragmented extracellular matrix)
The extracellular matrix is a material that fills the gaps between at least some cells in the cell structure. The extracellular matrix may be an aggregate of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. The extracellular matrix molecule may be a substance that exists outside the cell in an organism. The extracellular matrix is preferably a biocompatible material that is the same molecule as the molecule existing in the living body. As the extracellular matrix molecule, any substance can be used as long as it does not adversely affect the growth of cells and the formation of cell aggregates. Examples of the extracellular matrix molecule include, but are not limited to, elastin, collagen, laminin, fibronectin, vitronectin, tenascin, entactin, fibrillin, proteoglycan and the like. As the extracellular matrix, these may be used alone or in combination. The extracellular matrix may contain, for example, elastin and may be elastin. When the extracellular matrix is elastin, elastin functions as a scaffold for cell adhesion, further promoting the formation of three-dimensional cell structures. The extracellular matrix in the present embodiment is preferably a substance existing outside the animal cell, that is, an extracellular matrix of an animal. The extracellular matrix molecule may be a variant or variant of the above-mentioned extracellular matrix molecule as long as it does not adversely affect the growth of cells and the formation of cell aggregates, and is a polypeptide such as a chemically synthesized peptide. You may.

The extracellular matrix is a sequence represented by Gly-Val-Gly-Val-Pro characteristic of elastin (hereinafter also referred to as "GVGVP sequence", SEQ ID NO: 1) and / or Val-Gly-Val-Ala-Pro-Gly. It may have the sequence represented by (SEQ ID NO: 2 hereinafter, also referred to as “VGVAPG sequence”), and may have a repetition of these sequences.

When the extracellular matrix has a GVGVP sequence, improved elasticity and core selvation are promoted. In the extracellular matrix having a repeating GVGVP sequence, the proportion of the GVGVP sequence may be 80% or more, preferably 95% or more of the total amino acid sequence. In addition, one molecule of extracellular matrix may contain two or more GVGVP sequences.

Regarding the VGVAPG sequence, the function of inducing cell migration, proliferation, etc. has been reported. In the extracellular matrix having a repeating VGVAPG sequence, the proportion of the VGVAPG sequence may be 80% or more, preferably 95% or more of the total amino acid sequence. In addition, one molecule of extracellular matrix may contain two or more VGVAPG sequences.

Examples of elastin include vascular-derived elastin, lung-derived elastin, and skin-derived elastin. From the viewpoint of using it as a blood vessel wall model, it is preferable to use elastin derived from blood vessels. Further, from the viewpoint of using it as a blood vessel wall model having a blood vessel-related disease (for example, arteriosclerosis), it is preferable to use elastin derived from the aorta. Further, as elastin, either water-insoluble elastin or water-soluble elastin may be used, but it is preferable to use water-insoluble elastin.

Examples of collagen include fibrotic collagen and non-fibrotic collagen. The fibrous collagen means collagen which is a main component of collagen fibers, and specific examples thereof include type I collagen, type II collagen, and type III collagen. Examples of non-fibrotic collagen include type IV collagen.

Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan.

The extracellular matrix may contain at least one selected from the group consisting of elastin, collagen, laminin and fibronectin, preferably containing elastin. Elastin is preferably vascularly derived elastin. As the elastin derived from blood vessels, commercially available elastin may be used, and specific examples thereof include elastin derived from human aorta manufactured by Elastin Products Company.

When the extracellular matrix uses a plurality of types of mixed extracellular matrix such as type I and type III collagen, it may be separated into a single type of extracellular matrix by purification. For example, salt is added to a solution containing multiple types of mixed extracellular matrix, and the extracellular matrix of multiple types is separated by centrifugation, and the precipitate or supernatant is collected to obtain the desired type. Only extracellular matrix can be obtained. The obtained extracellular matrix of the desired type can be further salt-removed by dialysis, and may be further lyophilized after dialysis.

The extracellular matrix may be an animal-derived extracellular matrix. Examples of animal species from which the extracellular matrix is derived include, but are not limited to, humans, pigs, and cattle. As the extracellular matrix, components derived from one kind of animal may be used, or components derived from a plurality of kinds of animals may be used in combination. The animal species from which the extracellular matrix is derived may be the same as or different from the origin of the cells that are three-dimensionally organized.

Examples of the shape of the extracellular matrix include fibrous form. Fibrous means a shape composed of a filamentous extracellular matrix or a shape composed of a filamentous extracellular matrix cross-linked between molecules. At least a portion of the extracellular matrix may be fibrous. The shape of the extracellular matrix is the shape of a mass of extracellular matrix (aggregate of extracellular matrix) observed when observed under a microscope, and the extracellular matrix preferably has an average diameter and / or an average length described later. It has the size of. In the fibrous extracellular matrix, fine filaments (fibrils) formed by aggregating a plurality of filamentous extracellular matrix molecules, filaments formed by further aggregating fibrils, and these filaments are solved. Includes delicate ones. Including an extracellular matrix having a fibrous shape, for example, in the fibrous extracellular matrix, the GVGVP sequence (SEQ ID NO: 1) and the VGVAPG sequence (SEQ ID NO: 2) are conserved without being destroyed, and the cells are preserved. It can function even more effectively as a scaffolding material for adhesion.

"Fragmentation" means making the extracellular matrix aggregate smaller in size. The fragmented extracellular matrix (also referred to as "fragmented extracellular matrix") may include a defibrated extracellular matrix. The defibrated extracellular matrix is obtained by defibrating the above-mentioned extracellular matrix by applying a physical force. For example, defibration is performed under conditions that do not break the bonds within the extracellular matrix molecule.

The fragmented extracellular matrix may contain at least a portion of the defibrated extracellular matrix. Further, the fragmented extracellular matrix may consist only of the defibrated extracellular matrix. That is, the fragmented extracellular matrix may be a defibrated extracellular matrix. The defibrated extracellular matrix preferably contains defibrated elastin (defibrated elastin). The defibrated elastin preferably maintains a triple helix structure derived from elastin. The defibrated elastin may be a component that partially maintains the triple helix structure derived from elastin. Fragmented elastin can facilitate contact with cells in an aqueous medium and promote the formation of cell structures by dispersing it in an aqueous medium.

The average length of the fragmented extracellular matrix may be 100 nm or more and 400 μm or less, and may be 100 nm or more and 200 μm or less. In one embodiment, the average length of the fragmented extracellular matrix may be 5 μm or more and 400 μm or less, 10 μm or more and 400 μm or less, and 22 μm or more and 400 μm or less from the viewpoint of facilitating the formation of thick tissue. It may be 100 μm or more and 400 μm or less. In other embodiments, the average length of the fragmented extracellular matrix may be 100 μm or less, and may be 50 μm or less, from the viewpoint of facilitating tissue formation and further improving redispersibility. It may be 30 μm or less, 15 μm or less, 10 μm or less, 1 μm or less, and 100 nm or more. Of the entire fragmented extracellular matrix, it is preferable that the average length of most of the fragmented extracellular matrix is within the above numerical range. Specifically, it is preferable that the average length of 95% of the entire fragmented extracellular matrix is within the above numerical range. The fragmented extracellular matrix is preferably fragmented elastin having an average length within the above range, and more preferably defibrated elastin having an average length within the above range.

The average diameter of the fragmented extracellular matrix may be, for example, 20 nm or more and 30 μm or less, and may be 20 nm or more and 10 μm or less.

The content of the total fragmented extracellular matrix in the cell structure is, for example, 0.33% by mass or more, 0.5% by mass or more, or 5% by mass based on the total mass of the fragmented extracellular matrix and cells. That may be the above. The content of the fragmented extracellular matrix in the cell structure is, for example, 90% by mass or less, 80% by mass or less, 70% by mass or less, and 60% by mass based on the total mass of the fragmented extracellular matrix and cells. % Or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, or 1% by mass or less. The content of the fragmented extracellular matrix in the cell structure may be, for example, 0.33% by mass or more and 90% by mass or less based on the total mass of the fragmented extracellular matrix and cells. It may be 5% by mass or more and 90% by mass or less, 1.0% by mass or more and 90% by mass or less, 5% by mass or more and 90% by mass or less, and 5% by mass or more and 40% by mass or less. It may be there.

Since the above-mentioned average length and average diameter ranges are optimized from the viewpoint of tissue formation, it is desirable that the above-mentioned average length and average diameter range be within the above-mentioned average length or average diameter range at the stage of use for tissue formation.

The average length and diameter of the fragmented extracellular matrix can be determined by measuring each fragmented extracellular matrix with an optical microscope and performing image analysis. In the present specification, the "average length" means the average value of the lengths of the measured samples in the longitudinal direction, and the "average diameter" means the average value of the lengths of the measured samples in the direction orthogonal to the longitudinal direction. means.

The method for fragmenting the extracellular matrix is not particularly limited and may be fragmented by applying a physical force. The extracellular matrix fragmented by the application of physical force, unlike enzymatic treatment, usually has the same molecular structure as before fragmentation (the molecular structure is maintained). The method of fragmenting the extracellular matrix may be, for example, a method of finely crushing the massive extracellular matrix. The extracellular matrix may be fragmented in the solid phase or in an aqueous medium. For example, the extracellular matrix may be fragmented by applying a physical force such as an ultrasonic homogenizer, a stirring homogenizer, and a high-pressure homogenizer. When a stirring homogenizer is used, the extracellular matrix may be homogenized as it is, or it may be homogenized in an aqueous medium such as physiological saline. In addition, the average diameter of the fragmented extracellular matrix can be adjusted by adjusting the homogenizing time, the number of times, and the like. For example, homogenizing a dispersion dispersed in an aqueous medium containing extracellular matrix (eg, elastin) and then optionally incubating in an aqueous medium at 10 ° C. or lower (eg, 4 ° C. ± 1 ° C.). Be done. Examples of homogenization include homogenization using a homogenizer, stirring, applying pressure, and the like, and stirring may be performed using a stirrer. The aqueous medium may be pure water or an aqueous solution such as an aqueous solution containing phosphoric acid (eg, PBS buffer). Further, the time for incubating at 10 ° C. or lower (for example, 4 ° C. ± 1 ° C.) after homogenization may be, for example, 6 hours or longer, 12 hours or longer, 24 hours or longer, 48 hours or longer, or 72 hours or longer. .. The aqueous medium for homogenization (first aqueous medium) and the aqueous medium for culturing the cells may be the same or different. Also, the aqueous medium may or may not contain salts.

When the extracellular matrix is fragmented in an aqueous medium, the fragmented extracellular matrix comprises, for example, the step of fragmenting the extracellular matrix in an aqueous medium and the fragmented extracellular matrix and the aqueous medium. It can be produced by a method including a step of removing an aqueous medium from a liquid (removal step). The removal step may be carried out by, for example, a freeze-drying method. "Removing the aqueous medium" does not mean that no water is attached to the fragmented extracellular matrix components, but can be reached by common sense by the above-mentioned general drying method. It means that there is not enough water to adhere to it.

At least a portion of the fragmented extracellular matrix may be cross-linked or intramolecularly. The fragmented extracellular matrix may be cross-linked within the molecules that make up the fragmented extracellular matrix, or may be cross-linked between the molecules that make up the fragmented extracellular matrix.

Examples of the method for cross-linking include physical cross-linking by applying heat, ultraviolet rays, radiation and the like, chemical cross-linking by a cross-linking agent, an enzymatic reaction and the like, but the method is not particularly limited. The cross-linking (physical cross-linking and chemical cross-linking) may be a cross-linking via a covalent bond.

In fragmented elastin, crosslinks may be formed between elastin molecules (triple helix structure) or between elastin fibrils formed by elastin molecules. The cross-linking may be thermal cross-linking (thermal cross-linking). Thermal cross-linking can be carried out, for example, by performing heat treatment under reduced pressure using a vacuum pump. When thermally cross-linking an elastin molecule, the defibrated elastin may be cross-linked by forming a peptide bond (-NH-CO-) with the same amino group of the elastin molecule or the carboxy group of another elastin molecule. ..

The fragmented extracellular matrix can also be cross-linked by using a cross-linking agent. The cross-linking agent may be, for example, one capable of cross-linking a carboxyl group and an amino group, or one capable of cross-linking amino groups with each other. As the cross-linking agent, for example, aldehyde-based, carbodiimide-based, epoxide-based and imidazole-based cross-linking agents are preferable from the viewpoint of economy, safety and operability, and specifically, glutaraldehyde and 1-ethyl-3- (3). Examples thereof include water-soluble carbodiimides such as −dimethylaminopropyl) carbodiimide / hydrochloride and 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide / sulfonate.

The degree of cross-linking can be appropriately selected depending on the type of fragmented extracellular matrix, the means for cross-linking, and the like. The degree of cross-linking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and may be 30% or less, 20% or less, or 15% or less.

When the amino group in the extracellular matrix molecule is used for cross-linking, the degree of cross-linking can be quantified based on the TNBS method described in Non-Patent Document 2 and the like. The degree of cross-linking by the TNBS method may be within the above range. The degree of cross-linking by the TNBS method is the ratio of the amino groups used for cross-linking among the amino groups of the extracellular matrix.

The degree of cross-linking may be calculated by quantifying the carboxyl group. For example, in the case of an extracellular matrix that is insoluble in water, it may be quantified by the TBO (toluidine blue O) method. The degree of cross-linking by the TBO method may be within the above range.

The content of the extracellular matrix in the cell structure is 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, based on the dry weight of the cell structure. 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass, 15 It may be 90% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30. It may be 1% by mass or less, 20% by mass or less, or 15% by mass or less. The content of the extracellular matrix in the cell structure may be 0.01 to 90% by mass, based on the dry weight of the cell structure, and may be 10 to 90% by mass, 10 to 80% by mass, or 10 to 70% by mass. %, 10 to 60% by mass, 1 to 50% by mass, 10 to 50% by mass, 10 to 30% by mass, or 20 to 30% by mass.

Here, the "extracellular matrix in the cell structure" means the extracellular matrix constituting the cell structure, and may be derived from the endogenous extracellular matrix or is derived from the exogenous extracellular matrix. You may.

By "endogenous extracellular matrix" is meant extracellular matrix produced by extracellular matrix-producing cells. Examples of extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts. The endogenous extracellular matrix may be fibrotic or non-fibrotic.

"Extrinsic extracellular matrix" means an extracellular matrix supplied from the outside. The cell structure according to the present embodiment includes a fragmented extracellular matrix which is an exogenous extracellular matrix. The exogenous extracellular matrix may be derived from the same or different animal species as the endogenous extracellular matrix. Examples of the animal species from which it is derived include humans, pigs, and cattle. The exogenous extracellular matrix may also be an artificial extracellular matrix.

When the extracellular matrix is elastin, the exogenous extracellular matrix is also referred to as "exogenous elastin", and "extrinsic elastin", which means externally supplied elastin, is formed by multiple elastin molecules. It is an aggregate of elastin molecules. The extrinsic elastin is preferably vascular-derived elastin, more preferably aortic-derived elastin. As elastin, commercially available elastin may be used, and specific examples thereof include elastin derived from human aorta manufactured by Elastin Products Company.

In the exogenous extracellular matrix, the animal species from which it is derived may differ from the cell. In addition, when the cells contain extracellular matrix-producing cells, in the exogenous extracellular matrix, the animal species from which they are derived may be different from the extracellular matrix-producing cells. That is, the exogenous extracellular matrix may be a heterologous extracellular matrix.

That is, when the cell structure contains an endogenous extracellular matrix and a fragmented extracellular matrix, the content of the extracellular matrix constituting the cell structure is determined by the endogenous extracellular matrix and the fragmented extracellular matrix. Means the total amount of. The extracellular matrix content can be calculated from the volume of the obtained cell structure and the mass of the decellularized cell structure.

For example, when the extracellular matrix contained in the cell structure is collagen, as a method for quantifying the amount of collagen in the cell structure, for example, the following method for quantifying hydroxyproline can be mentioned. A sample is prepared by mixing hydrochloric acid (HCl) with a lysate in which a cell structure is dissolved, incubating at a high temperature for a predetermined time, returning to room temperature, and diluting the centrifuged supernatant to a predetermined concentration. The hydroxyproline standard solution is treated in the same manner as the sample, and then diluted stepwise to prepare the standard. Each of the sample and the standard is subjected to a predetermined treatment with a hydroxyproline assay buffer and a detection reagent, and the absorbance at 570 nm is measured. The amount of collagen is calculated by comparing the absorbance of the sample with the standard. The cell structure may be directly suspended in high-concentration hydrochloric acid, and the dissolved solution may be centrifuged to recover the supernatant and used for quantifying the collagen component. Further, the cell structure to be lysed may be in a state of being recovered from the culture solution, or may be lysed in a state of being dried after the recovery to remove the liquid component. However, when quantifying the collagen component by dissolving the cell structure as it is collected from the culture solution, the cells are affected by the medium component absorbed by the cell structure and the remaining influence of the medium due to the problem of the experimental procedure. Since the measured value of the structure weight is expected to vary, it is preferable to use the weight after drying as a reference from the viewpoint of stably measuring the weight of the tissue and the amount of collagen per unit weight.

More specifically, as a method for quantifying the amount of collagen, the following methods can be mentioned, for example.

(Sample preparation)
The entire amount of the lyophilized cell structure is mixed with 6 mol / L HCl, incubated in a heat block at 95 ° C. for 20 hours or more, and then returned to room temperature. After centrifuging at 13000 g for 10 minutes, the supernatant of the sample solution is collected. A sample is prepared by appropriately diluting with 6 mol / L HCl so that the result falls within the range of the calibration curve in the measurement described later, and then diluting 200 μL with 100 μL of ultrapure water. 35 μL of sample is used.

(Standard preparation)
125 μL of standard solution (1200 μg / mL in acetic acid) and 125 μL of 12 mol / l HCl are added to the screw cap tube, mixed, incubated in a heat block at 95 ° C. for 20 hours, and then returned to room temperature. After centrifugation at 13000 g for 10 minutes, the supernatant is diluted with ultrapure water to prepare S1 of 300 μg / mL, and S1 is diluted stepwise to S2 (200 μg / mL), S3 (100 μg / mL), S4. (50 μg / mL), S5 (25 μg / mL), S6 (12.5 μg / mL), S7 (6.25 μg / mL) are prepared. Also prepare S8 (0 μg / mL) containing only 90 μL of 4 mol / l HCl.

(A assay)
35 μL of standard and sample are added to the plate (with QuickZyme Total Collagen Assay Kit, QuickZyme Biosciences), respectively. Add 75 μL of assay buffer (included in the kit above) to each well. Close the plate with a seal and incubate at room temperature for 20 minutes shaking. Remove the seal and add 75 μL of reagent reagent (reagent A: B = 30 μL: 45 μL, included in the kit above) to each well. Close the plate with a seal, mix the solutions with shaking and incubate at 60 ° C. for 60 minutes. Allow to cool sufficiently on ice, remove the seal and measure the absorbance at 570 nm. The amount of collagen is calculated by comparing the absorbance of the sample with the standard.

Elastin in the cell structure may be defined by its area ratio or volume ratio. "Defined by area ratio or volume ratio" means that elastin in a cell structure can be distinguished from other tissue constituents by, for example, a known staining method (for example, immunostaining using an anti-elastin antibody). After that, it means to calculate the ratio of the elastin-existing region to the entire cell structure by using macroscopic observation, various microscopes, image analysis software, and the like. When the area ratio is specified, the area ratio is not limited by what cross section or surface in the cell structure, but for example, when the cell structure is a sphere or the like, a cross-sectional view passing through a substantially central portion thereof. May be specified by.

For example, when elastin in a cell structure is defined by an area ratio, the ratio of the area is 0.01 to 99% and 1 to 99% based on the total area of the cell structure. It is preferably 5 to 90%, preferably 7 to 90%, preferably 20 to 90%, and more preferably 50 to 90%. "Elastin in the cell structure" is as described above. The ratio of the area of elastin constituting the cell structure means the ratio of the combined area of endogenous elastin and exogenous elastin. The ratio of the area of elastin can be calculated, for example, as the ratio of the area of stained elastin to the total area of the cross section of the obtained cell structure stained and passing through the substantially central portion of the cell structure. ..

The cell structure preferably has a trypsin concentration of 0.25%, a temperature of 37 ° C., a pH of 7.4, and a residual rate of 70% or more after trypsin treatment at a reaction time of 15 minutes, preferably 80% or more. It is more preferable, and 90% or more is even more preferable. Such a cell structure is stable because it is unlikely to be decomposed by an enzyme during or after culturing. The residual rate can be calculated, for example, from the mass of the cell structure before and after the trypsin treatment.

The cell structure may have a residual rate of 70% or more after elastase treatment at a concentration of elastase of 0.25%, a temperature of 37 ° C., a pH of 7.4, and a reaction time of 15 minutes, and may be 80% or more. It is more preferably present, and even more preferably 90% or more. Such a cell structure is stable because it is unlikely to be decomposed by an enzyme during or after culturing.

The thickness of the cell structure is preferably 10 μm or more, more preferably 100 μm or more, and even more preferably 1000 μm or more. Such a cell structure has a structure closer to that of a living tissue, and is suitable as a substitute for an experimental animal and a material for transplantation. The upper limit of the thickness of the cell structure is not particularly limited, but may be, for example, 10 mm or less, 3 mm or less, 2 mm or less, or 1.5 mm or less. It may be 1 mm or less.

Here, the "thickness of the cell structure" means the distance between both ends in the direction perpendicular to the main surface when the cell structure is in the shape of a sheet or a rectangular parallelepiped. When the main surface is uneven, the thickness means the distance at the thinnest part of the main surface.

When the cell structure is spherical or substantially spherical, it means the diameter thereof. Furthermore, when the cell structure is ellipsoidal or substantially ellipsoidal, it means its minor axis. When the cell structure is substantially spherical or approximately ellipsoidal and the surface is uneven, the thickness is the shortest distance between two points where a straight line passing through the center of gravity of the cell structure and the surface intersect. Means the distance of.

(Vascular endothelial cell layer)
The vascular endothelial cell layer in the cell structure according to the present embodiment contains vascular endothelial cells.

Endothelial cells mean flat cells that make up the surface of the lumen of blood vessels. The vascular endothelial cells may be, for example, human umbilical vein-derived vascular endothelial cells (HUVEC). The vascular endothelial cells constituting the cell structure may be primary cells (primary vascular endothelial cells) collected from the umbilical vein or umbilical artery of an animal (for example, human), and are cells obtained by culturing the primary cells. It may be a cultured cell line in which a primary cell is established, or a cell artificially differentiated from a stem cell. Examples of the cultured cell line include a cultured cell line of product model number T0056 manufactured by Applied Biological Materials. Examples of stem cells to be differentiated include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The vascular endothelial cells contained in the cell structure according to the present embodiment may be non-cancerous cells.

The ratio (X ₂ / X ₀ x 100) of the number of vascular endothelial cells (X ₂ ) to the total number of cells (X ₀ ) in the cell structure is 5% or more, 10% or more, 12% or more, 14% or more. , 15% or more, 20% or more, or 25% or more, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. May be. The ratio (X ₂ / X ₀ × 100) of the number of vascular endothelial cells (X ₂ ) to the total number of cells (X ₀ ) in the cell structure is 5% or more from the viewpoint of being more suitable as a vascular wall tissue. It may be 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, 10% or more and 25% or less, or 12% or more and 20% or less.

The vascular endothelial cell layer may further contain a fragmented extracellular matrix.

The thickness of the vascular endothelial cell layer is preferably 5 μm to 30 μm, more preferably 5 μm to 15 μm. The upper limit of the thickness of the vascular endothelial cell layer is not particularly limited, but may be, for example, 30 μm or less, 20 μm or less, or 10 μm or less. From the viewpoint of using as a blood vessel wall model having a disease (for example, arteriosclerosis) in which the vascular endothelial cell layer becomes thicker than usual, the thickness of the vascular endothelial cell layer is preferably 5 μm to 15 μm.

Here, the "thickness of the cell layer" means a distance in a direction perpendicular to the main surface when the cell structure is in the form of a sheet or a rectangular parallelepiped.

When the cell structure is spherical or substantially spherical, it means the distance in the radial direction thereof. Furthermore, when the cell structure is ellipsoidal or substantially ellipsoidal, it means the distance in the minor axis direction.

The ratio of the number of smooth muscle cells to vascular endothelial cells (smooth muscle cells / vascular endothelial cells) may be 9/1 to 99/1, 50/50 to 80/20, or 20. It may be / 80 to 50/50, or 10/90 to 50/50.

In the cell structure of the present embodiment, the smooth muscle cell layer and the vascular endothelial cell layer are in contact with each other at least in part.

The cell structure of this embodiment contains immune cells.

Immune cells are cells that have the ability to recognize antigens and react specifically, and are involved in immunity. The immunocyte may be, for example, a macrophage, its progenitor cells monocytes, dendritic cells, or lymphocytes such as T cells, B cells and natural killer (NK) cells, and may be macrophages and / or monocytes. There may be. The immune cell may be, for example, a human peripheral blood monocyte cell line (THP-1). The immune cells contained in the cell structure may be primary cells (primary immune cells) collected from peripheral blood or lymph fluid of an animal (for example, human), or may be cells obtained by culturing primary cells. It may be a cultured cell line in which a primary cell is established, or it may be a cell artificially differentiated from a stem cell. Examples of the cultured cell line include a cultured cell line having the cell number JCRB0112 of the JCRB cell bank. Examples of stem cells to be differentiated include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The immune cell contained in the cell structure according to the present embodiment may be a non-cancerous cell.

The ratio of the number of immune cells (X ₂ ) to the total number of cells (X _{0 ) in the cell structure (X 3} / X ₀ × 100) is 5% or more, 10% or more, 12% or more, 14% or more, It may be 15% or more, 20% or more, or 25% or more, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. It may be there. The ratio of the number of immune cells (X ₃ ) to the total number of cells (X _{0 ) in the cell structure (X 3} / X ₀ × 100) is even more suitable as a vascular wall tissue as a vascular wall model with vascular disease. From this viewpoint, it may be 5% or more and 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, 10% or more and 25% or less, or 12% or more and 20% or less.

Immune cells may be contained on the surface of the cell structure, may be contained inside the cell structure, and may be contained both on the surface and inside. In addition, the immune cells may be contained in the smooth muscle cell layer or the vascular endothelial cell layer, and may be contained in the surface and / or inside of the smooth muscle cell layer or the vascular endothelial cell layer.

In order to confirm that the cell structure contains immune cells, for example, it can be determined whether the expression of cell adhesion molecules is increased as compared with the cell structure containing no immune cells. Examples of cell adhesion molecules include ICAM-1 (intercellular adhesion molecule-1), which is known to be involved in the binding of activated lymphocytes to vascular endothelial cells. Also, for example, whether the expression of CD11b, which suggests the adhesion of monocytes, and / or the expression of CD14, which suggests the differentiation of monocytes into macrophages, is increased as compared with the cell structure containing no immune cells. You can also judge. The co-localization of CD11b and CD14 means that the cell structure contains macrophages. For example, it is preferable that 90% or more of CD11b per unit area (for example, 1 mm ^{2) in the cell structure or each layer is co-localized with CD14.} Similarly, for example, by analyzing the expression of cell adhesion molecules, CD11b, CD14, etc. in each layer of the smooth muscle cell layer or the vascular endothelial cell layer, it is possible to determine whether immune cells are contained on the surface and / or inside. can.

Immune cells may contain lipoproteins internally. Examples of lipoproteins include low-density lipoprotein (LDL), remnant and Lp (a). The lipoprotein may be a denatured lipoprotein. Modified lipoproteins include, for example, oxidized lipoproteins, glycated lipoproteins and acetylated lipoproteins. The denatured lipoprotein may be, for example, oxidized LDL, glycated LDL and acetylated LDL. Further, for example, the immune cell may be a macrophage and may take up acetylated LDL.

In this embodiment, the cells may include cells other than smooth muscle cells, vascular endothelial cells and immune cells. Other cells may be, for example, mature somatic cells or undifferentiated cells such as stem cells. Specific examples of somatic cells include nerve cells, lymphatic endothelial cells, fibroblasts, epithelial cells, myocardial cells, pancreatic islet cells, osteoocytes, alveolar epithelial cells, spleen cells and the like. Examples of stem cells include ES cells, iPS cells, mesenchymal stem cells and the like. The other cell may be a normal cell, or may be a cell in which any cell function is enhanced or suppressed, such as a cancer cell. A "cancer cell" is a cell derived from a somatic cell that has acquired infinite proliferative capacity.

The origin of smooth muscle cells, vascular endothelial cells, immune cells or other cells contained in the cell structure is not particularly limited, but for example, humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats and the like. It may be a cell derived from a mammalian animal of.

Since the cell structure according to the present embodiment has a structure similar to that of a vascular tissue of a living body and contains immune cells, it can be used as a vascular wall model having a vascular disease, for example, an arteriosclerotic vascular wall model.

[Method for manufacturing cell structure containing immune cells]
The method for producing a cell structure containing immune cells according to the present embodiment is to mix smooth muscle cells and fragmented extracellular matrix in an aqueous medium and incubate them to form a smooth muscle cell layer, and the above-mentioned smoothing. A step of preparing a cell structure including contacting a vascular endothelial cell with a muscle cell layer and incubating it to form a vascular endothelial cell layer in contact with the smooth muscle cell layer.
The step of bringing the pro-inflammatory agent into contact with the cell structure and
It includes a step of bringing immune cells into contact with the cell structure.

"Aqueous medium" means a liquid containing water as an essential constituent. The aqueous medium is not particularly limited as long as the fragmented extracellular matrix and cells can exist stably. For example, physiological saline such as phosphate buffered saline (PBS), Dulvecco's Modified Eagle's medium (DMEM), smooth muscle cell medium (SMCGM2), vascular endothelial cell medium (EGM2), monosphere medium ( Examples include liquid media such as RPMI). The liquid medium may be a mixed medium in which two types of media are mixed. From the viewpoint of reducing the load on the cells, the aqueous medium is preferably a liquid medium. The medium preferably contains a medium for smooth muscle cells when proliferating smooth muscle cells, and preferably contains a medium for vascular endothelial cells when proliferating vascular endothelial cells, and proliferates immune cells. In some cases, it is preferable to include a medium for immune cells. When the medium contains a medium for smooth muscle cells and a medium for vascular endothelial cells, the ratio (medium for smooth muscle cells: medium for vascular endothelial cells) is preferably 1 to 5: 1-5. When the medium contains a medium for smooth muscle cells, a medium for vascular endothelial cells, and a medium for immune cells, the ratio (medium for smooth muscle cells: medium for vascular endothelial cells: medium for immune cells) is 1 to 1. It is preferably 5: 1 to 5: 1 to 5.

The incubator (support) used for incubating is not particularly limited, and may be, for example, a well insert, a low-adhesion plate, or a plate having a bottom shape such as a U-shape or a V-shape. The cells may be cultured while being adhered to the support, the cells may be cultured without being adhered to the support, or the cells may be separated from the support and cultured in the middle of the culture. When the cells are cultured without adhering to the support, or when the cells are separated from the support during culturing and cultured, they have a bottom shape such as a U-shape or a V-shape that inhibits the adhesion of the cells to the support. It is preferable to use a plate or a low adsorption plate.

The method of incubation is not particularly limited, and can be carried out by a suitable method depending on the type of cells. For example, the incubation temperature may be 20 ° C to 40 ° C or 30 ° C to 37 ° C. The pH of the medium at the time of incubation may be 6-8 or 7.2-7.4. The incubation time may be 1 day to 2 weeks or 1 week to 2 weeks for the entire production of the cell structure.

[Preparation of cell structure]
Smooth muscle cells and fragmented extracellular matrix are mixed and incubated in an aqueous medium to form a smooth muscle cell layer, and vascular endothelial cells are contacted and incubated with the smooth muscle cell layer to incubate and smooth. A cell structure is prepared by a step including forming a vascular endothelial cell layer in contact with a muscle cell layer.

(Formation of smooth muscle cell layer)
Preparation of the cell structure comprises mixing and incubating smooth muscle cells and fragmented extracellular matrix in an aqueous medium to form a smooth muscle cell layer.

By dispersing the fragmented extracellular matrix in an aqueous medium, it becomes easier to contact cells in the aqueous medium and can promote the formation of cell structures. A cell suspension is obtained by mixing the cells and the fragmented extracellular matrix in an aqueous medium and dispersing the cells and the fragmented extracellular matrix.

Examples of the method of mixing smooth muscle cells and fragmented extracellular matrix in an aqueous medium include a method of mixing an aqueous medium containing a fragmented extracellular matrix and an aqueous medium containing smooth muscle cells, and fragmented extracellular matrix. A method of adding smooth muscle cells to an aqueous medium containing a matrix, a method of adding an aqueous medium containing a fragmented extracellular matrix to a culture medium containing smooth muscle cells, a method of adding a smooth muscle to an aqueous medium containing a fragmented extracellular matrix. Examples thereof include, but are not limited to, a method of adding cells and a method of adding fragmented extracellular matrix and smooth muscle cells to an aqueous medium prepared in advance. In addition, the fragmented extracellular matrix and smooth muscle cells may be stirred by pipetting or the like in an aqueous medium.

The aqueous medium (second aqueous medium) for mixing smooth muscle cells and the fragmented extracellular matrix in the aqueous medium is the aqueous medium (first aqueous medium) for homogenizing the above-mentioned fragmented extracellular matrix. ) May be the same or different, but it is preferable to include a medium for smooth muscle cells. Further, even if the aqueous medium for mixing the smooth muscle cells and the fragmented extracellular matrix contains a medium for smooth muscle cells and a medium for vascular endothelial cells, the medium for smooth muscle cells, the medium for vascular endothelial cells, and the medium for vascular endothelial cells It may contain a medium for immune cells.

The concentration of the fragmented extracellular matrix in the aqueous medium that mixes the smooth muscle cells and the fragmented extracellular matrix depends on the shape, thickness, size of the incubator, etc. of the target cell structure and smooth muscle cell layer. Can be decided as appropriate. For example, the concentration of the fragmented extracellular matrix in the aqueous medium may be 0.1 to 90% by weight or 1 to 30% by weight.

The amount of fragmented extracellular matrix to be mixed with the smooth muscle cells, i.e. the amount contained in the cell suspension, for example, with respect to 1.0 × ¹⁰ 6 cells of smooth muscle cells, 0.1-100 mg, 0. It may be 5 to 50 mg, 0.8 to 25 mg, 1.0 to 10 mg, 1.0 to 5.0 mg, 1.0 to 2.0 mg, or 1.0 to 1.8 mg, and 0.7 mg or more. It may be 1.1 mg or more, 1.2 mg or more, 1.3 mg or more or 1.4 mg or more, 7.0 mg or less, 3.0 mg or less, 2.3 mg or less, 1.8 mg or less, 1.7 mg or less, 1 It may be 0.6 mg or less or 1.5 mg or less. The mass ratio of the fragmented extracellular matrix to the smooth muscle cells (fragmented extracellular matrix / smooth muscle cells) is preferably 1/1 to 1000/1, and is 9/1 to 900/1. More preferably, it is more preferably 10/1 to 500/1.

It may include adding fibrinogen and / or thrombin after mixing the smooth muscle cells with the fragmented extracellular matrix and prior to incubation. When both fibrinogen and thrombin are added, for example, fibrinogen and thrombin may be added at the same time, or fibrinogen may be added first and then thrombin may be added. The timing of adding fibrinogen and / or trombine is not particularly limited, and for example, it may be added to an aqueous medium containing smooth muscle cells and fragmented extracellular matrix, and contains smooth muscle cells and fragmented extracellular matrix. Trombin may be added after adding fibrinogen to the aqueous medium. The addition of fibrinogen and / or thrombin can suppress possible shrinkage during incubation and facilitate control of the shape and size of cell structures. Further, since the mixed solution of smooth muscle cells and fragmented extracellular matrix can be gelled, the mixed solution can be easily peeled off from the incubator after being dropped on the incubator (support).

In addition, the production method of the present embodiment includes a step of mixing smooth muscle cells and fragmented extracellular matrix and then applying an external force to accumulate smooth muscle cells and fragmented extracellular matrix before incubating. But it may be. By performing such a step, the distribution of fragmented extracellular matrix and smooth muscle cells in the cell structure becomes more uniform. Specific methods are not particularly limited, and examples thereof include a method of centrifuging an aqueous medium containing extracellular matrix and smooth muscle cells. The conditions for centrifugation can be set as appropriate, and examples thereof include centrifugation at 200 to 3000 rpm for 1 to 10 minutes.

By incubating the smooth muscle cells mixed in an aqueous medium and the fragmented extracellular matrix, the smooth muscle cells in contact with the fragmented extracellular matrix are cultured to form a smooth muscle cell layer.

The method for incubating an aqueous medium (cell suspension) containing extracellular matrix and smooth muscle cells is not particularly limited, but for example, the incubation temperature may be 20 ° C to 40 ° C, and 30 ° C to 37 ° C. It may be ° C. The pH of the aqueous medium in which the smooth muscle cells and the fragmented extracellular matrix are mixed may be 6 to 8 or 7.2 to 7.4. The incubation time may be, for example, 1 day to 2 weeks, 2 days to 1 week, and 2 days to 5 days.

The incubator (support) used for incubating the cell suspension is not particularly limited, and may be, for example, a well insert, a low-adhesion plate, or a plate having a bottom shape such as a U-shape or a V-shape. The cells may be cultured while being adhered to the support, the cells may be cultured without being adhered to the support, or the cells may be separated from the support and cultured in the middle of the culture. When the cells are cultured without adhering to the support, or when the cells are separated from the support during culturing and cultured, a U-shaped or V-shaped bottom surface that inhibits the adhesion of the cells to the support is formed. It is preferable to use a plate having a culture or a low adsorption plate.

By incubating (culturing), smooth muscle cells contacted with the fragmented extracellular matrix in the cell suspension are cultured. Incubation may include the cells in contact with the fragmented extracellular matrix without adhering to the support. As a result, it is possible to produce a cell structure that is aggregated in a mass without being adhered to the support. If the cells in contact with the fragmented extracellular matrix are attached to the support, the fragmented extracellular matrix may include pulling the contacted cells away from the support. When cells in contact with the fragmented extracellular matrix in culture do not adhere to the support from the beginning, the cells are cultured as they are to produce a cell structure that aggregates in a mass without adhering to the support. can do.

The method of pulling the cells into contact with the fragmented extracellular matrix from the support is not particularly limited, for example, using a low-adhesion support and pulling the cells away from the support by adding a culture medium. The cells may be physically separated from the support by using an instrument or the like, or the cells may be separated from the support by applying vibration, and the cells may be supported by responding to a stimulus such as heat and light. A support coated with a functional material that breaks the bond between the body and the cells may be used, and the cells may be separated from the support by stimulating the support. As the culture medium when the cells are separated from the support by adding the culture medium, the medium or the like exemplified in the present specification can be used.

In culturing, as a method of culturing cells in contact with the fragmented extracellular matrix from the beginning without adhering to the support, for example, a suspension containing cells in contact with the fragmented extracellular matrix is used. After gelling the cells and gently dropping them into the culture medium for culturing, and fixing the shape of the cells in contact with the fragmented extracellular matrix in a high-viscosity solvent to some extent, removing only the solvent and culturing the culture vessel. There is a method to move to.

In the culture, the timing of pulling the cells in contact with the fragmented extracellular matrix from the support is not particularly limited, and may be, for example, 1 to 7 days from the start of the incubation, and 1 to 24 hours. It may go for 1 to 60 minutes, 5 to 30 minutes, or 10 to 20 minutes.

The incubation period after the cells contacted by the fragmented extracellular matrix from the support are separated is not particularly limited, and may be 1 day or more, 1 day to 21 days, and 3 days to 14 days. It can be a day, 7 to 14 days.

The cell density in the medium in the incubation (culture) can be appropriately determined according to the shape and thickness of the target cell structure and the smooth muscle cell layer, the size of the incubator, and the like. For example, cell density in the culture medium in the culture ^may be 1 to 10 8 cells / ^mL, may be 10 ³ ~10 7 cells / mL.

The thickness of the smooth muscle cell layer in the cell structure is preferably 20 μm to 500 μm, more preferably 50 μm to 200 μm. The upper limit of the thickness of the smooth muscle cell layer is not particularly limited, but may be, for example, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less. May be good. From the viewpoint of using as a blood vessel wall model having vascular disease (for example, arteriosclerosis), the thickness of the smooth muscle cell layer is preferably 50 μm to 150 μm. It is preferable that the smooth muscle cell layer is finally formed to the above-mentioned preferable thickness by incubation.

The production method according to the present embodiment may further include heating the extracellular matrix before fragmenting the extracellular matrix to crosslink at least a part of the extracellular matrix, or after fragmentation. And it may be provided that the extracellular matrix is heated to cross at least a portion of the extracellular matrix before mixing with smooth muscle cells.

In cross-linking, the temperature (heating temperature) and time (heating time) when heating the extracellular matrix can be appropriately determined. The heating temperature may be, for example, 100 ° C. or higher, 200 ° C. or lower, or 220 ° C. or lower. Specifically, the heating temperature is, for example, 100 ° C., 110 ° C., 120 ° C., 130 ° C., 140 ° C., 150 ° C., 160 ° C., 170 ° C., 180 ° C., 190 ° C., 200 ° C., 220 ° C. and the like. good. The heating time (time to hold at the above heating temperature) can be appropriately set depending on the heating temperature. The heating time may be, for example, 6 hours or more and 72 hours or less, more preferably 24 hours or more and 48 hours or less when heating at 100 ° C. to 200 ° C. In the cross-linking, heating may be performed in the absence of a solvent, or heating may be performed under reduced pressure conditions.

The production method according to the present embodiment may include drying the fragmented extracellular matrix after fragmentation.

In drying, the defibrated extracellular matrix is dried. Drying may be carried out by, for example, a freeze-drying method. After defibration, drying is performed to remove the aqueous medium from the solution containing the fragmented extracellular matrix component and the aqueous medium. Removal of the aqueous medium does not mean that no water is attached to the fragmented extracellular matrix and can be sensiblely reached by the general drying techniques described above. It means that there is no moisture attached to the extent.

(Formation of vascular endothelial cell layer)
Preparation of the cell structure comprises contacting the vascular endothelial cells with the smooth muscle cell layer and incubating them to form a vascular endothelial cell layer in contact with the smooth muscle cell layer.

Examples of the method of contacting the vascular endothelial cells with the smooth muscle cell layer include a method of adding vascular endothelial cells to the aqueous medium forming the smooth muscle cell layer, and a method of including the vascular endothelial cells in the aqueous medium forming the smooth muscle cell layer. Examples include, but are not limited to, a method of adding an aqueous medium and a method of removing the aqueous medium once after forming the smooth muscle cell layer and then adding an aqueous medium containing vascular endothelial cells to the smooth muscle cell layer. .. Further, the aqueous medium containing vascular endothelial cells may be an aqueous medium containing a fragmented extracellular matrix. When the fragmented extracellular matrix is contained, it is preferable to use a cell suspension in which vascular endothelial cells and the fragmented extracellular matrix are mixed in an aqueous medium.

When the aqueous medium containing the vascular endothelial cells contains the fragmented extracellular matrix, the concentration of the fragmented extracellular matrix in the aqueous medium is determined by the shape, thickness, and thickness of the target cell structure and vascular endothelial cell layer. It can be appropriately determined according to the size of the incubator and the like. For example, the concentration of the fragmented extracellular matrix in the aqueous medium may be 0.1 to 90% by weight or 1 to 30% by weight.

The amount of fragmented extracellular matrix mixed with the vascular endothelial cells may be 0.1 to 100 mg or 1 to 50 mg ^{per 1 × 10 5 cells of the vascular endothelial cells.} The mass ratio of the fragmented extracellular matrix to the vascular endothelial cells (fragmented extracellular matrix / vascular endothelial cells) may be 1/1 to 1000/1, and may be 9/1 to 900/1. It may be 10/1 to 500/1.

The aqueous medium for incubating vascular endothelial cells preferably contains a medium for vascular endothelial cells. When an aqueous medium containing vascular endothelial cells (third aqueous medium) is added separately, the aqueous medium (first aqueous medium) for homogenizing the above-mentioned fragmented extracellular matrix and smooth muscle cells and fragments. It may be the same as or different from any of the aqueous mediums (second aqueous mediums) for mixing with the extracellular matrix, but preferably contains a medium for vascular endothelial cells.

The steps of adding fibrinogen and / or thrombin in the formation of smooth muscle cell layers and applying external force prior to incubation to accumulate cells and fragmented extracellular matrix apply similarly to the formation of vascular endothelial cell layers. be able to.

By contacting and incubating the vascular endothelial cells with the smooth muscle cell layer, the vascular endothelial cells are cultured and the vascular endothelial cell layer in contact with the smooth muscle cell layer is formed.

The method of bringing the vascular endothelial cells into contact with the smooth muscle cell layer and incubating the cells is not particularly limited, and for example, the incubation temperature may be 20 ° C. to 40 ° C. or 30 ° C. to 37 ° C. The pH of the aqueous medium containing the vascular endothelial cells may be 6-8 or 7.2-7.4. The incubation time may be, for example, 5 days to 2 weeks, 1 week to 2 weeks, 5 days to 8 days.

The cell density in the medium in the incubation (culture) can be appropriately determined according to the shape and thickness of the target cell structure and the vascular endothelial cell layer, the size of the incubator, and the like. For example, cell density in the culture medium in the culture ^may be 1 to 10 8 cells / ^mL, may be 10 ³ ~10 7 cells / mL.

The thickness of the vascular endothelial cell layer is preferably 5 μm to 30 μm, more preferably 5 μm to 15 μm. The upper limit of the thickness of the vascular endothelial cell layer is not particularly limited, but may be, for example, 30 μm or less, 20 μm or less, or 10 μm or less. From the viewpoint of using as a blood vessel wall model having a disease (for example, arteriosclerosis) in which the vascular endothelial cell layer becomes thicker than usual, the thickness of the vascular endothelial cell layer is preferably 5 μm to 15 μm. It is preferred that the incubation finally forms the vascular endothelial cell layer to the preferred thickness described above.

By the production step described above, a stable cell structure in which cells are uniformly distributed can be obtained.

The cell structure produced by the production method according to the present embodiment preferably has a shrinkage rate of 20% or less, more preferably 15% or less, and further preferably 10% or less during culturing. .. The shrinkage rate can be calculated by the following formula, for example. In the formula, L1 indicates the length of the longest portion of the cell structure on the first day after culturing, and L3 indicates the length of the corresponding portion of the cell structure on the third day after culturing.

Shrinkage rate (%) = {(L1-L3) / L1} x 100
In the above example, the contraction rate is calculated from the cell structure on the first day after culturing and the cell tissue on the third day after culturing, but the cell structure at any time during the culturing period including the end time of culturing. It may be calculated from. For example, it may be calculated from the cell structure on the first day after culturing and the cell structure on the second day after culturing, or it may be calculated from the cell structure on the first day after culturing and the cell structure on the fifth day after culturing. It may be calculated from the cell structure on the first day after culturing and the cell structure on the eighth day after culturing.

[Step of contacting the cell structure with the pro-inflammatory agent and step of contacting the immune cells]
The method for producing a cell structure containing immune cells according to the present embodiment includes a step of bringing an inflammation promoter into contact with the prepared cell structure and a step of bringing immune cells into contact with the cell structure.

By contacting the immune cells with the cell structure to which the pro-inflammatory agent has been contacted, the cell structure can contain the immune cells. Contact of an pro-inflammatory agent with cells such as smooth muscle cells promotes the expression of cell adhesion factors and inflammatory cytokines, resulting in immune cell adhesion and cell layer infiltration in endothelial cells. Immune cells may be contained on the surface of the cell structure, may be contained inside the cell structure, and may be contained both on the surface and inside. In addition, the immune cells may be contained in the smooth muscle cell layer or the vascular endothelial cell layer, and may be contained in the surface and / or inside of the smooth muscle cell layer or the vascular endothelial cell layer.

(Step of contacting the pro-inflammatory agent with the cell structure)
The pro-inflammatory agent is not particularly limited as long as the cell structure can contain immune cells, and examples thereof include factors that induce arteriosclerosis, specifically, Angiotensin II and lipoprotein. The lipoprotein may be low density lipoprotein (LDL), acetylated lipoprotein, or acetylated LDL.

As a method of bringing the inflammation promoter into contact with the cell structure, a method of adding it in advance to an aqueous medium for producing the cell structure (for example, an aqueous medium for forming a smooth muscle cell layer), or a method for producing the cell structure was prepared. A method of adding an inflammatory agent to an aqueous medium (for example, an aqueous medium for forming a vascular endothelial cell layer), a method of adding an aqueous medium containing an inflammatory agent to an aqueous medium in which a cell structure is prepared, a method of adding a cell structure. Examples thereof include, but are not limited to, a method of removing the aqueous medium once after preparation and adding an aqueous medium containing an inflammation promoter.

When an aqueous medium containing an inflammation promoter (fourth aqueous medium) is added separately, the aqueous medium (first aqueous medium) for homogenizing the above-mentioned fragmented extracellular matrix, smooth muscle cells and fragments. It may be the same as or different from either the aqueous medium (second aqueous medium) for mixing with the extracellular matrix and the aqueous medium containing vascular endothelial cells (third aqueous medium).

The concentration of the pro-inflammatory agent is not particularly limited as long as the cell structure can contain immune cells, but the higher the concentration of the pro-inflammatory agent, the thicker the vascular endothelial cell layer tends to be. It can be appropriately set according to the thickness of the vascular endothelial cell layer. For example, when the pro-inflammatory agent is Angiotensin II, 0.1 μM to 5 μM with respect to the aqueous medium containing the cell structure, and when the pro-inflammatory agent is lipoprotein, the aqueous medium containing the cell structure is used. On the other hand, the concentration can be 200 to 500 μg / mL. By increasing the concentration of the pro-inflammatory agent, it is possible to produce a cell structure suitable for use as a blood vessel wall model having a disease (for example, arteriosclerosis) in which the vascular endothelial cell layer becomes thicker than usual.

(Step of contacting immune cells with the cell structure)
Immune cells are as described above in [Cell Structure]. Immune cells may contain the above lipoproteins internally. The lipoprotein may be low density lipoprotein (LDL), acetylated lipoprotein, or acetylated LDL. For example, the immune cell may be a macrophage and may take up acetylated LDL.

Examples of the method of bringing the immune cells into contact with the cell structure include a method of adding the immune cells to an aqueous medium in which the cell structure is prepared (for example, an aqueous medium for forming a vascular endothelial cell layer), and an aqueous medium in which the cell structure is prepared. Examples thereof include, but are not limited to, a method of adding an aqueous medium containing immune cells to the medium, and a method of removing the aqueous medium once after preparing the cell structure and adding the aqueous medium containing immune cells.

The aqueous medium for contacting the immune cells with the cell structure preferably contains a medium for immune cells. When an aqueous medium containing immune cells (fifth aqueous medium) is added separately, the aqueous medium (first aqueous medium) for homogenizing the above-mentioned fragmented extracellular matrix, smooth muscle cells and fragmentation Either an aqueous medium (second aqueous medium) for mixing with the extracellular matrix, an aqueous medium containing vascular endothelial cells (third aqueous medium), or an aqueous medium containing an inflammation promoter (fourth aqueous medium). It may be the same as or different from the above, but it is preferable to include a medium for immune cells.

Incubation may be performed after contacting the immune cells with the cell structure. The method of bringing the immune cells into contact with the cell structure and incubating the cells is not particularly limited, and for example, the incubation temperature may be 20 ° C. to 40 ° C. or 30 ° C. to 37 ° C. The pH of the aqueous medium containing the immune cells may be 6-8 or 7.2-7.4. The incubation time may be, for example, 2 days to 2 weeks, 2 days to 1 week, and 2 days to 5 days.

The step of contacting the immune cells with the cell structure preferably comprises contacting the immune cells with the vascular endothelial cell layer.

The method for confirming that the cell structure contains immune cells can be carried out in the same manner as the method described above in [Cell structure].

As the specific embodiment in the production method according to the present embodiment, the specific embodiment in the cell structure described above can be applied without limitation.

[Method of evaluating the effect of a drug using a cell structure]
As described above, since the cell structure according to the present embodiment has a structure closer to the vascular tissue of a living body and contains immune cells, it should be used as a vascular wall model having a vascular disease, for example, an arteriosclerotic vascular wall model. Can be done. Therefore, the cell structure according to the present embodiment can be used to evaluate the effect of the drug or screen the drug.

As an embodiment of the present invention, a method for evaluating the effect of a drug using the above-mentioned cell structure is provided.

The evaluation method according to the present embodiment preferably includes an administration step of administering the drug to the cell structure and an evaluation step of evaluating the effect of the drug by changes in the cell structure to which the drug is administered.

The drug for which the effect is evaluated is not particularly limited, but from the viewpoint that the characteristics of the cell structure according to the present embodiment are utilized, a drug related to vascular disease (a drug for treating or preventing), particularly a drug related to arteriosclerosis. Is preferable. Examples of the drug related to arteriosclerosis include an arteriosclerosis inhibitor and an arteriosclerosis promoter. Examples of the arteriosclerosis inhibitor include high density lipoprotein (HDL), apolipoprotein AI, statins, PCSK9 inhibitors and the like. Examples of the arteriosclerosis promoter include denatured LDL, remnant, Lp (a), Angiotensin II and the like.

The administration of the drug to the cell structure may be carried out by using a medium containing the above drug as the medium for culturing the cell structure, or by adding the above drug to the medium for culturing the cell structure. You may.

The cell structure to which the drug is administered may be a cell structure that has been cultured for 1 day or longer, a cell structure that has been cultured for 5 days or longer, or a cell structure that has been cultured for 6 days or longer. , It may be a cell structure cultured for 7 days or more, a cell structure cultured for a longer number of days, or a cell structure cultured for a period of 7 days or more and 14 days or less.

In the evaluation step, the drug efficacy is evaluated by changes in the cell structure to which the drug is administered. Examples of the change as an index of evaluation include a change in the vascular endothelial cell layer and an adhesion amount of immune cells. The change in the vascular endothelial cell layer may be, for example, a change in the thickness of the vascular endothelial cell layer or a change in elasticity. The medicinal effect may be evaluated based on the change of only one kind of the above-mentioned index, or may be evaluated based on the change of two or more kinds of the above-mentioned index. Changes in the cell structure may be evaluated by qualitative comparison or quantitative comparison.

The evaluation step can be performed, for example, by comparing the vascular endothelial cell layer in the cell structure to which the drug has been administered with the vascular endothelial cell layer in the cell structure to which the drug has not been administered. Further, for example, it can be carried out by comparing the adhesion amount of immune cells in the cell structure to which the drug has been administered with the adhesion amount of immune cells in the cell structure to which the drug has not been administered.

In the evaluation step, for example, when the thickness of the vascular endothelial cell layer in the drug-administered cell structure is smaller than that in the vascular endothelial cell layer in the drug-administered cell structure, vascular disease is caused. It is evaluated to be effective as a drug for treating or preventing, and if the thickness of the vascular endothelial cell layer in the cell structure to which the drug is administered is large or unchanged, it is not effective as a drug for treating or preventing vascular disease. May be evaluated as. Further, for example, a drug that promotes arteriosclerosis when the thickness of the vascular endothelial cell layer in the cell structure to which the drug is administered is larger than that in the vascular endothelial cell layer in the cell structure to which the drug is not administered. If the thickness of the vascular endothelial cell layer in the cell structure to which the drug is administered is small or unchanged, it may be evaluated as ineffective as a drug that promotes arteriosclerosis.

Further, in the evaluation step, for example, when the amount of immune cells adhered to the drug-administered cell structure is smaller than the amount of immune cells adhered to the drug-administered cell structure, vascular disease. Is evaluated to be effective as a drug for treating or preventing vascular disease, and if the amount of adhesion of immune cells in the cell structure to which the drug is administered is large or unchanged, it is not effective as a drug for treating or preventing vascular disease. You may evaluate it. Further, for example, when the amount of immune cells adhered to the cell structure to which the drug was administered is larger than the amount of immune cells adhered to the cell structure to which the drug was not administered, as a drug that promotes arteriosclerosis. It may be evaluated as effective, and if the amount of immune cells adhered to the cell structure to which the drug is administered is small or unchanged, it may be evaluated as ineffective as a drug that promotes arteriosclerosis.

As another embodiment of the present invention, in a cell structure having a smooth muscle cell layer containing smooth muscle cells and a fragmented extracellular matrix, and a vascular endothelial cell layer containing vascular endothelial cells.
The process of contacting the pro-inflammatory agent,
The process of contacting immune cells,
Also provided is a method for evaluating the effect of a drug, which comprises an administration step of administering the drug to a cell structure and an evaluation step of evaluating the effect of the drug based on changes in the cell structure to which the drug is administered.

As the specific mode and the like in the evaluation method according to the present embodiment, the above-mentioned specific mode and the like in the cell structure and the method for producing the same can be applied without limitation.

Further, as an embodiment of the present invention, a method for screening a drug using a cell structure is provided. According to this embodiment, for example, an agent for treating or preventing a vascular disease can be effectively selected.

The method for screening a drug using a cell structure may include a step of selecting a drug evaluated to be effective as a drug for treating or preventing a vascular disease in the evaluation step in the above evaluation method.

The changes that serve as an index for evaluation are as described above in the above evaluation method. The medicinal effect may be evaluated based on the change of only one kind of the above-mentioned index, or may be evaluated based on the change of two or more kinds of the above-mentioned index. Changes in the cell structure may be evaluated by qualitative comparison or quantitative comparison.

Further, the screening method includes, for example, a step of measuring the thickness of the vascular endothelial cell layer in the cell structure to which the drug is administered, and a step of measuring the thickness of the vascular endothelial cell layer.
The thickness of the vascular endothelial cell layer in the drug-treated cell structure is compared with the thickness of the vascular endothelial cell layer in the drug-treated cell structure, and the thickness of the vascular endothelial cell layer in the drug-treated cell structure is compared. Includes, or includes, a step of selecting the drug as a candidate for a drug to treat or prevent vascular disease when the thickness is small.
The step of measuring the elasticity of the vascular endothelial cell layer in the cell structure to which the drug was administered, and
The elasticity of the vascular endothelial cell layer in the drug-treated cell structure is compared with the elasticity of the vascular endothelial cell layer in the drug-treated cell structure, and the elasticity of the vascular endothelial cell layer in the drug-treated cell structure. Includes, or includes, a step of selecting the drug as a candidate for a drug to treat or prevent vascular disease when is small.
The process of measuring the amount of immune cell adhesion in the cell structure to which the drug was administered, and
When the amount of adhesion of immune cells in the cell structure to which the drug was administered is compared with the amount of adhesion of immune cells in the cell structure to which the drug was not administered, and the amount of adhesion of immune cells in the cell structure to which the drug was administered is small. Can include a step of selecting the above-mentioned drug as a candidate substance of a drug for treating or preventing vascular disease.

The candidate substance is not particularly limited, but is preferably a drug related to vascular disease, particularly a drug related to arteriosclerosis, from the viewpoint of utilizing the characteristics of the cell structure according to the present embodiment. Examples of the drug related to arteriosclerosis include an arteriosclerosis inhibitor and an arteriosclerosis promoter. Examples of the arteriosclerosis inhibitor include high density lipoprotein (HDL), apolipoprotein AI, statins, PCSK9 inhibitors and the like. Examples of the arteriosclerosis promoter include denatured LDL, remnant, Lp (a), Angiotensin II and the like.

The comparison of the thickness of the vascular endothelial cell layer in the cell structure may be performed multiple times. That is, the comparison of the thickness of the vascular endothelial cell layer in the cell structure may be performed a plurality of times at predetermined intervals after the administration of the drug.

The elasticity of the vascular endothelial cell layer in the cell structure may be compared multiple times. That is, the comparison of the elasticity of the vascular endothelial cell layer in the cell structure may be performed a plurality of times at predetermined intervals after the administration of the drug.

Moreover, the comparison of the adhesion amount of immune cells in the cell structure may be performed a plurality of times. That is, the comparison of the adhesion amount of immune cells in the cell structure may be performed a plurality of times at predetermined intervals after administration of the drug.

The administration of the drug can be carried out in the same manner as the evaluation method described above. Further, as the cell structure or the like to be used, the above-mentioned one can be used.

As the specific aspect of the screening method according to the present embodiment, the above-mentioned cell structure, the method for producing the same, the specific aspect of the evaluation method, and the like can be applied without limitation.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

The following cells and medium were prepared to prepare the cell structure.
(cell)
Human umbilical vein-derived endothelial cells (HUVEC): Lonza human umbilical artery-derived smooth muscle cells (UASMC): Lonza human monocyte cells: THP-1 of JCRB cell bank
(Culture medium)
Medium for smooth muscle cells: Product name SMCGM2, Product code C-22062, Medium for vascular endothelium manufactured by Promocell: Product name EGM2MV, Product code CC-3202, Medium for monocytes manufactured by Lonza: Product name RPMI, Product code 30264 85, made by Nacalai Tesque

<Test Example 1: Preparation of fragmented elastin>
Fragmented elastin (hereinafter referred to as "elastin microfiber") is obtained by suspending 50 mg of bovine blood vessel-derived elastin (product name SB87, manufactured by Elastin Products Company) in 5 mL of Milli-Q and homogenizing at room temperature for 6 minutes using a stirring homogenizer. (EMF) ”) was obtained. The average diameter of the obtained fragmented elastin was 1.3 ± 0.4 μm, and the average length (length) was 9.2 ± 4.8 μm.

<Test Example 2: Preparation of cell structure>
A 24-well insert (UASMC) of 50 μL (fragmented elastin, equivalent to 0.5 mg) of the dispersion containing fragmented elastin obtained in Test Example 1 and 8 × 10 ⁵ cells of human umbilical artery-derived smooth muscle cells (UASMC) was inserted. It was added to Corning). After centrifuging at 1100 rpm for 5 minutes, the cells were cultured at 37 ° C. for 2 days to construct a smooth muscle cell layer. Further, on the constructed smooth muscle cell layer, 2 × 10 ⁵ human umbilical vein-derived vascular endothelial cells (HUVEC) were suspended in a medium and deposited on the smooth muscle cell layer by natural sedimentation. A cell structure having a vascular endothelial cell layer on a smooth muscle cell layer was prepared by culturing at 37 ° C. for 7 days. The prepared cell structure was fixed with 10% paraformaldehyde (PFA), sliced, stained with hematoxylin and eosin (HE), and immunostained with an anti-CD31 antibody. It was confirmed that the cell structure contained a high concentration of elastin microfiber and had a smooth muscle cell layer and a vascular endothelial cell layer. It was also confirmed that when the cell structure was observed from above, the upper surface of the cell structure was uniformly covered with vascular endothelial cells.

<Test Example 3: Angiotensin II treatment (analysis of monocyte cell adhesion and differentiation)>
Angiotensin II, which is one of the inducers of arteriosclerosis, promotes the expression of cell adhesion factor and inflammatory cytokine by contact with smooth muscle cells, and causes adhesion of immune cells and cell layer infiltration in endothelial cells. I know. Therefore, the effect of Angiotensin II on the cell structure of Test Example 2 was analyzed.

Specifically, in Test Example 2, after preparing a cell structure having a vascular endothelial cell layer on a smooth muscle cell layer, 5 × 10 ⁴ human monocyte cells were further formed on the vascular endothelial cell layer. (THP-1) was suspended in a medium, deposited on the vascular endothelial cell layer by natural sedimentation, and cultured at 37 ° C. for 7 days. When human monocytic cells were added and cultured, the mass ratio of the smooth muscle cell medium, the vascular endothelial cell medium, and the monocytic cell medium was 1: 1: 1, and further, Angiotensin II (Ang II) ( The culture was carried out separately in the case where the product code 4001-V (manufactured by Peptide Institute) was not included and the case where the product code was 1 μM. After fixing with methanol, it was sliced and immunostained with an anti-CD11b antibody (product code 301305, manufactured by BioLegend) and an anti-CD14 antibody (product code 367101, manufactured by BioLegend).

The immunostained photograph is shown in FIG. 1, and the graph in which the comparison is made by the fluorescence area is shown in FIG. Expression of CD11b suggests monocyte adhesion and expression of CD14 suggests monocyte differentiation. CD11b and CD14 are strongly expressed and CD11b and CD14 are co-localized in the cell structure cultured in the medium containing Angiotensin II as compared with the cell structure cultured in the medium containing Angiotensin II. It was shown to be (“Ang II +” in FIGS. 1 and 2). These results suggest that Angiotensin II promotes adhesion of human monocyte cells (THP-1) and differentiation into macrophages.

<Test Example 4: Angiotensin II treatment (expression analysis of cell adhesion molecule)>
Anti-CD31 antibody (anti-CD31 antibody) was used to obtain a cell structure cultured in a medium containing Angiotensin II prepared in the same manner as in Test Example 3 except that human monocyte cells were cultured on the vascular endothelial cell layer for 5 days. Immunostaining was performed with product code M0823 (manufactured by DAKO) and an anti-CD54 antibody (product code 116101, manufactured by BioLegend). An immunostained photograph (observed from above) is shown in FIG. 3, and a graph comparing the fluorescence areas of the anti-CD54 antibody by immunostaining is shown in FIG. The expression of the anti-CD54 antibody suggests the expression of ICAM, which is a kind of cell adhesion molecule. In FIG. 3, the part shown in white shows the fluorescence due to the anti-CD54 antibody, and the part shown in black shows the fluorescence due to the anti-CD31 antibody. From these results, strong expression of anti-CD54 antibody was confirmed in the cell structure, suggesting strong expression of ICAM.

In addition, strong expression of anti-CD54 antibody was confirmed in the tissues prepared in the same manner as the cell structure except for the elastin microfibers (“without EMF” in FIGS. 3 and 4), but on the elastin microfibers. Anti-CD54 antibody in tissues prepared by culturing vascular endothelial cells in (Fig. 3 and FIG. 4 “without SMC”) and in tissues prepared only with vascular endothelial cells (“HUVEC only” in FIGS. 3 and 4). The expression of was not confirmed. Similar to the cell structure, all the tissues were prepared using a medium having a mass ratio of 1: 1: 1 for smooth muscle cell medium: vascular endothelial cell medium: monocytic cell medium. These results also suggested that a smooth muscle cell layer was required for ICAM expression.

FIG. 5 shows a graph comparing the immunostaining fluorescence area of the anti-CD54 antibody between a cell structure cultured in a medium containing different concentrations of Angiotensin II and a tissue prepared only of vascular endothelial cells (“HUVEC”). show. FIG. 5A shows a comparison of fluorescence areas, and FIG. 5B shows a comparison of fluorescence area ratios when the fluorescence area is 1.0 when Angiotensin II is not included. In tissues prepared only with vascular endothelial cells, the fluorescence area did not increase even if the concentration of Angiotensin II was increased, but in the cell structure, when the concentration of Angiotensin II was 0.1 μM or more, the fluorescence area increased. Increased fluorescence was observed. These results suggest that the action of Angiotensin II may increase the expression of ICAM in the cell structure.

<Test Example 5: Acetylated Low Density Lipoprotein (LDL) Treatment>
It is known that acetylated low-density lipoprotein (LDL), which is one of the inducers of arteriosclerosis, promotes cell proliferation and infiltration of immune cells and is taken up by macrophages. Therefore, the effect of acetylated low-density lipoprotein on the cell structure of Test Example 2 was analyzed.

Specifically, in Test Example 2, after preparing a cell structure having a vascular endothelial cell layer on a smooth muscle cell layer, 5 × 10 ⁴ human monocyte cells were further formed on the vascular endothelial cell layer. (THP-1) was suspended in a medium, deposited on the vascular endothelial cell layer by natural sedimentation, and cultured at 37 ° C. for 3 days. Then, human-derived acetylated low-density lipoprotein (distributed from the National Cerebral and Cardiovascular Research Center) was added to the medium at a concentration of 0 to 400 μg / mL, and further cultured at 37 ° C. for 5 days. The prepared cell structure was fixed with 10% paraformaldehyde (PFA), sliced, and stained with hematoxylin and eosin (HE). The results are shown in FIG. The concentration in the upper left of each photograph indicates the concentration of acetylated low-density lipoprotein. As the concentration of acetylated low-density lipoprotein increased, the thickness of the endothelial cell layer tended to increase.

The graph of FIG. 7 shows the change in the thickness (μm) of the endothelial cell layer of the cell structure with respect to the concentration of acetylated low-density lipoprotein. In tissues made only of vascular endothelial cells (“HUVEC only”), the thickness of the endothelial cell layer did not change due to changes in the concentration of acetylated low-density lipoprotein (LDL concentration / μg / mL), but cells. In the structure, the thickness of the endothelial cell layer tended to increase as the concentration of acetylated low-density lipoprotein increased.

Further, FIG. 8 shows a photograph of the prepared cell structure immunostained with an anti-CD31 antibody (product code M0823, manufactured by DAKO) and an anti-α-SMA antibody (product code 904601, manufactured by BioLegend). "HUVE C only" indicates the result of using a tissue prepared only of vascular endothelial cells. FIG. 8A shows the results of immunostaining with the anti-CD31 antibody, and FIG. 8B shows the results of immunostaining with the anti-α-SMA antibody. The concentration shown in the upper left of each photograph indicates the concentration of acetylated low-density lipoprotein. From FIG. 8 (a), it was found that vascular endothelial cells (HUVEC, the portion represented by black) are widely distributed in the endothelial cell layer. It was also shown from FIG. 8 (b) that smooth muscle cells (SMC, portion represented in black) were not present in the endothelial cell layer. The endothelial cell layer of the cell structure is composed of endothelial cells and THP-1. It was suggested that the smooth muscle cell layer promotes absorption and infiltration of THP-1 to thicken the endothelial cell layer in response to acetylated low-density lipoprotein.

<Test Example 6: High Density Lipoprotein (HDL) Treatment>
High-density lipoprotein (HDL) is the major lipoprotein in the blood and is composed of phospholipids and apolipoproteins, which mediate cholesterol transport in the body. In addition, high-density lipoprotein is known to have many anti-arteriosclerosis effects such as cholesterol extraction ability, antioxidant effect, anti-inflammatory effect, and suppression of endothelial cell activity. Therefore, the effect of high-density lipoprotein on the cell structure of Test Example 4 was analyzed. ^{The cell structure of Test Example 4 is obtained by depositing 5 × 10 4} human monocyte cells (THP-1) on a cell structure having a vascular endothelial cell layer on a smooth muscle cell layer and culturing it.

Specifically, human monocyte cells (THP-1) were deposited and cultured at 37 ° C. for 3 days, and then human-derived acetylated low-density lipoprotein (distributed from the National Cerebral and Cardiovascular Research Center). 200 μg / mL and human-derived high-density lipoprotein (distributed from the National Cerebral and Cardiovascular Research Center) 0 to 2000 μg / mL were added to the medium, and the cells were further cultured at 37 ° C. for 5 days. The prepared cell structure was fixed with 10% paraformaldehyde (PFA), sliced, and immunostained with an anti-CD31 antibody (product code M0823, manufactured by DAKO). A graph showing the change in the thickness of the endothelial cell layer with respect to the protein concentration is shown in FIG. The inside of FIG. 9 separated by a dotted line corresponds to the endothelial cell layer (the part shown in white is the endothelial cell), and the concentration on each photograph indicates the concentration of high-density lipoprotein. The sample (0 μg / mL) to which high-density lipoprotein was not added had the thickest endothelial cell layer due to the influence of acetylated low-density lipoprotein, and the higher the concentration of high-density lipoprotein, the thicker the endothelial cell layer. There was a tendency to decrease.

Claims (12)

A method for producing a cell structure containing immune cells.
Smooth muscle cells and fragmented extracellular matrix are mixed and incubated in an aqueous medium to form a smooth muscle cell layer, and vascular endothelial cells are contacted and incubated with the smooth muscle cell layer to incubate the smooth muscle cell layer. Forming a vascular endothelial cell layer in contact with a muscle cell layer, and a process for producing a cell structure containing
The step of bringing the pro-inflammatory agent into contact with the cell structure and
A method comprising contacting an immune cell with the cell structure. The production method according to claim 1, wherein the step of contacting the immune cells with the cell structure comprises contacting the immune cells with the vascular endothelial cell layer. The production method according to claim 1 or 2, wherein the fragmented extracellular matrix has the sequence represented by SEQ ID NO: 1 and / or the sequence represented by SEQ ID NO: 2. The production method according to claim 3, wherein the fragmented extracellular matrix is elastin. The production method according to any one of claims 1 to 4, wherein the pro-inflammatory agent is Angiotensin II or lipoprotein. The production method according to claim 5, wherein the lipoprotein is a denatured lipoprotein. A smooth muscle cell layer containing smooth muscle cells and a fragmented extracellular matrix,
Has a vascular endothelial cell layer, which contains vascular endothelial cells,
The smooth muscle cell layer and the vascular endothelial cell layer are in contact with each other.
A cell structure that contains immune cells. The cell structure according to claim 7, wherein the smooth muscle cell layer or the vascular endothelial cell layer contains immune cells. The cell structure according to claim 7 or 8, wherein the immune cell contains a lipoprotein inside. A method for evaluating the effect of a drug using the cell structure according to claim 7 or 8. The administration step of administering the drug to the cell structure,
The evaluation method according to claim 10, further comprising an evaluation step of evaluating the effect of the drug based on changes in the cell structure to which the drug is administered. The evaluation method according to claim 10 or 11, wherein the agent is an arteriosclerosis inhibitor or an arteriosclerosis promoter.

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