阅读说明：本技术 含细胞外基质组合物及其制造方法、以及三维组织体及其制造方法 (Extracellular matrix-containing composition, method for producing same, three-dimensional tissue, and method for producing same ) 是由 北野史朗 入江新司 松崎典弥 于 2020-03-25 设计创作，主要内容包括：本发明涉及一种含细胞外基质组合物,其包含经片段化的细胞外基质成分和键合或吸附于经片段化的细胞外基质成分的化合物。(The present invention relates to an extracellular matrix-containing composition comprising a fragmented extracellular matrix component and a compound bonded or adsorbed to the fragmented extracellular matrix component.)

1. An extracellular matrix-containing composition comprising a fragmented extracellular matrix component and a compound bonded to or adsorbed to the fragmented extracellular matrix component.

2. The extracellular matrix-containing composition of claim 1,

the fragmented extracellular matrix components comprise fragmented collagen components.

3. The extracellular matrix-containing composition according to claim 1 or 2,

the average length of the fragmented extracellular matrix components is 100nm or more and 200 μm or less.

4. The extracellular matrix-containing composition according to any one of claims 1 to 3,

the compound is at least 1 selected from heparan sulfate, chondroitin sulfate and fibronectin.

5. A method for producing an extracellular matrix-containing composition, which comprises a step of bringing a fragmented extracellular matrix component into contact with a compound that can be bonded to or adsorbed to the fragmented extracellular matrix component.

6. The method for producing an extracellular matrix-containing composition according to claim 5,

the fragmented extracellular matrix components are obtained by fragmenting extracellular matrix components in an aqueous medium.

7. The method for producing an extracellular matrix-containing composition according to claim 5 or 6,

the fragmented extracellular matrix components comprise fragmented collagen components.

8. A method for manufacturing a three-dimensional structure, comprising:

a first step of bringing the extracellular matrix-containing composition according to any one of claims 1 to 4 into contact with cells in an aqueous medium; and

a second step of culturing the cells contacted with the extracellular matrix-containing composition.

9. The method for manufacturing a three-dimensional tissue body according to claim 8,

the method further comprises a step of sedimenting the fragmented extracellular matrix component, the compound bonded or adsorbed to the fragmented extracellular matrix component, and the cells in the aqueous medium after the first step and before the second step.

10. The method for producing a three-dimensional tissue body according to claim 8 or 9,

the first step is performed after a layer of the cells is formed in an aqueous medium.

11. The method for producing a three-dimensional structure according to any one of claims 8 to 10,

the cells comprise extracellular matrix producing cells.

12. The method for producing a three-dimensional structure according to any one of claims 8 to 11,

the cells comprise one or more cells selected from the group consisting of vascular endothelial cells, cancer cells, cardiac myocytes, smooth muscle cells, and epithelial cells.

13. The method for producing a three-dimensional tissue body according to any one of claims 8 to 12, wherein,

the total content of the fragmented extracellular matrix component and the compound is 10 mass% or more and 90 mass% or less based on the total mass of the fragmented extracellular matrix component, the compound, and the cells.

14. A three-dimensional tissue body comprising cells and the extracellular matrix-containing composition of any one of claims 1-4, wherein at least a portion of the cells are contacted with the fragmented extracellular matrix components.

Technical Field

The present invention relates to an extracellular matrix-containing composition and a method for producing the same, and a three-dimensional tissue and a method for producing the same.

Background

As a method for artificially producing a structure imitating a living tissue, for example, the following methods are known: a method for producing a three-dimensional tissue body by culturing a coated cell in which the whole surface of a cultured cell is coated with an adhesive film (patent document 1); a method for producing a three-dimensional tissue body (patent document 2) comprising a step of three-dimensionally arranging cells coated with a coating film containing collagen to form a three-dimensional tissue body; a method for producing a three-dimensional tissue body (patent document 3) comprising a step of forming coated cells in which a coating is formed on the surface of a cell, and a step of arranging the coated cells three-dimensionally, wherein the formation of the coated cells comprises a step of immersing the cells in a liquid containing a coating component, and a step of separating the immersed cells from the liquid containing the coating component by means of a liquid-permeable membrane; a method for producing a three-dimensional cell tissue (patent document 4) includes the steps of: the cells are mixed with a cationic substance and an extracellular matrix component to obtain a mixture, and the cells are collected from the obtained mixture to form a cell aggregate on a substrate.

The present inventors have also proposed a method for producing a three-dimensional tissue body having a high collagen concentration by bringing cells into contact with endogenous collagen, preferably further with fibrous exogenous collagen (patent document 5). Such a three-dimensional tissue body is expected to be used as a substitute for an experimental animal, a transplant material, and the like.

However, in vivo, extracellular matrices such as collagen interact to form tissues. In addition, it is suggested that extracellular matrix is bonded and/or adsorbed to a lower molecular compound for extracellular matrix interaction, and that the bonded and/or adsorbed compound plays an important role in the interaction of extracellular matrix (for example, non-patent documents 1 to 3).

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 2012 and 115254

Patent document 2: international publication No. 2015/072164

Patent document 3: international publication No. 2016/027853

Patent document 4: international publication No. 2017/146124

Patent document 5: international publication No. 2018/143286

Non-patent document

Non-patent document 1: m.c. erat et al, proc.natl.acad.sci.usa 2009,106,4195

Non-patent document 2: y. tatara et al, Glycobiology 2015,25,557.

Non-patent document 3: m.shawn et al, proc.natl.acad.sci.usa 1998,95,7275.

Disclosure of Invention

Problems to be solved by the invention

According to the method for producing a three-dimensional structure, although a three-dimensional structure can be easily produced by an artificial method, it is required to produce a three-dimensional structure that can more faithfully simulate the interaction of various compounds in an actual living body.

An object of one aspect of the present invention is to provide an extracellular matrix-containing composition capable of forming a three-dimensional tissue body closer to the state in a living body. Another object of the present invention is to provide a three-dimensional tissue body which is closer to the state in a living body, and a method for producing the same.

Means for solving the problems

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

[1] An extracellular matrix-containing composition comprising a fragmented extracellular matrix component and a compound bonded or adsorbed to the fragmented extracellular matrix component.

[2] The extracellular matrix-containing composition according to [1], wherein the fragmented extracellular matrix components comprise fragmented collagen components.

[3] The extracellular matrix-containing composition according to [1] or [2], wherein the average length of the fragmented extracellular matrix components is 100nm or more and 200 μm or less.

[4] The extracellular matrix-containing composition according to any one of [1] to [3], wherein the compound is at least 1 selected from the group consisting of heparan sulfate, chondroitin sulfate and fibronectin.

[5] A method for producing an extracellular matrix-containing composition, which comprises a step of bringing a fragmented extracellular matrix component into contact with a compound that can be bonded to or adsorbed to the fragmented extracellular matrix component.

[6] The method for producing an extracellular matrix-containing composition according to [5], wherein the fragmented extracellular matrix components are obtained by fragmenting extracellular matrix components in an aqueous medium.

[7] The method for producing an extracellular matrix-containing composition according to [5] or [6], wherein the fragmented extracellular matrix components comprise fragmented collagen components.

[8] A method for manufacturing a three-dimensional structure, comprising: a first step of bringing the extracellular matrix-containing composition according to any one of [1] to [4] into contact with cells in an aqueous medium; and a second step of culturing the cells contacted with the extracellular matrix-containing composition.

[9] The method of producing a three-dimensional tissue according to [8], which comprises a step of sedimenting the fragmented extracellular matrix component, the compound bonded or adsorbed to the fragmented extracellular matrix component, and the cells in the aqueous medium after the first step and before the second step.

[10] The method of producing a three-dimensional structure according to [8] or [9], wherein the first step is performed after a layer of cells is formed in an aqueous medium.

[11] The method for producing a three-dimensional tissue body according to any one of [8] to [10], wherein the cells comprise extracellular matrix-producing cells.

[12] The method for producing a three-dimensional tissue body according to any one of [8] to [11], wherein the cells include one or more cells selected from vascular endothelial cells, cancer cells, cardiac muscle cells, smooth muscle cells, and epithelial cells.

[13] The method for producing a three-dimensional tissue body according to any one of [8] to [12], wherein the total content of the fragmented extracellular matrix component and the compound is 10 mass% or more and 90 mass% or less based on the total mass of the fragmented extracellular matrix component, the compound, and the cells.

[14] A three-dimensional tissue body comprising cells and the extracellular matrix-containing composition according to any one of [1] to [4], wherein at least a part of the cells are contacted with the fragmented extracellular matrix components.

Effects of the invention

According to the present invention, an extracellular matrix-containing composition capable of forming a three-dimensional tissue body in a state closer to that in a living body can be provided. According to the present invention, a three-dimensional tissue body in a state close to that in a living body and a method for producing the same can be provided. By using the extracellular matrix-containing composition of the present invention, for example, a three-dimensional tissue body which mimics the presence of blood vessels and cells around blood vessels can be obtained. Furthermore, by using the extracellular matrix-containing composition of the present invention, a three-dimensional tissue body having a blood vessel density thicker than that of the conventional one can be obtained.

Drawings

Fig. 1 is a photomicrograph of a fibrillated collagen component having various compounds bonded or adsorbed thereto.

Fig. 2 is a photomicrograph of a defibrated collagen component bonded or adsorbed with fluorescein labeled chondroitin sulfate.

Fig. 3 is a photograph showing the observation result of a capillary vessel composed of a three-dimensional tissue body stained with CD 31.

Fig. 4 is a graph showing the measurement results of the diameter of the capillary vessel.

Fig. 5 is a photograph showing the observation results of capillaries composed of three-dimensional tissue bodies stained with Vimentin (Vimentin) and CD 31.

Fig. 6 is a photograph showing the observation result of a three-dimensional tissue body stained with Toluidine Blue (TB).

FIG. 7 is a graph showing the measurement results of the length of cell nuclei in a three-dimensional tissue body.

Fig. 8 is a photograph showing the observation result of a three-dimensional tissue stained with CD 31.

Detailed Description

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

< composition containing extracellular matrix >

The extracellular matrix-containing composition of the present embodiment includes a fragmented extracellular matrix component (fragmented extracellular matrix component) and a compound bonded or adsorbed to the fragmented extracellular matrix component.

The extracellular matrix-containing composition of the present embodiment can be suitably used as a scaffold material or the like for forming a three-dimensional tissue body.

The extracellular matrix component is a material that fills at least a part of intercellular spaces in a three-dimensional tissue body. An extracellular matrix component is an aggregate of extracellular matrix molecules formed from a plurality of extracellular matrix molecules. The extracellular matrix molecule may be a substance present outside the cell in an organism. Any extracellular matrix molecule may be used as long as it does not adversely affect the growth of cells and the formation of cell aggregates. Examples of extracellular matrix molecules include, but are not limited to, collagen, laminin, fibronectin, elastin, cell adhesin, entactin, fibrin, and proteoglycan. The extracellular matrix components may be used alone or in combination of 1. The extracellular matrix component may include or consist of collagen, for example. When the extracellular matrix component contains collagen, the collagen functions as a scaffold for cell adhesion, and further promotes the formation of a three-dimensional cellular structure. The extracellular matrix molecule may be a modified form or a mutant of the above-mentioned extracellular matrix molecule, or a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect the growth of cells or the formation of cell aggregates. The extracellular matrix molecule may have a repeating sequence represented by Gly-X-Y characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent an arbitrary amino acid residue. The plurality of Gly-X-Y may be the same or different. By having a repeating sequence represented by Gly-X-Y, the restriction on the arrangement of the molecular chains is small, and the function as a scaffold material is more excellent. In the extracellular matrix molecule having a repeating sequence represented by Gly-X-Y, the proportion of the sequence represented by Gly-X-Y may be 80% or more, preferably 95% or more, of the total amino acid sequence. In addition, the extracellular matrix molecule may be a polypeptide having an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). By having the RGD sequence, cell adhesion is further promoted, and is more suitable as a scaffold material. Examples of extracellular matrix molecules including a sequence represented by Gly-X-Y and an RGD sequence include collagen, fibronectin, laminin, cadherin, and the like.

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

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

The extracellular matrix component may comprise at least 1 selected from collagen, laminin and fibronectin, preferably collagen. The collagen is preferably fibrous collagen, more preferably type I collagen. As the fibrous collagen, commercially available collagen can be used, and specific examples thereof include type I collagen derived from pigskin manufactured by Ham corporation of japan.

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

The fragmented extracellular matrix components can be obtained by fragmenting the extracellular matrix components described above. "fragmentation" refers to the bringing together of extracellular matrix molecules into a smaller size. The fragmentation may be performed under conditions in which the bond in the extracellular matrix molecule is cleaved, or may be performed without cleaving the bond in the extracellular matrix molecule. The fragmented extracellular matrix components may include a component obtained by defibering the above extracellular matrix components by applying a physical force, that is, a defibered extracellular matrix component. Defibration is a means of fragmentation, and is carried out, for example, without cleaving bonds within extracellular matrix molecules.

The method for fragmenting the extracellular matrix component is not particularly limited. As a method for defibrating the extracellular matrix components, for example, the extracellular matrix components may be defibrated by applying physical force such as an ultrasonic homogenizer, a stirring homogenizer, or a high-pressure homogenizer. When the agitation type homogenizer is used, the extracellular matrix components may be directly homogenized, or may be homogenized in an aqueous medium such as physiological saline. Further, by adjusting the time, the number of times, etc. of the homogenization, it is also possible to obtain a defibrated extracellular matrix component of millimeter size or nanometer size. The defibrated extracellular matrix component may be obtained by defibrating by repeating freeze thawing.

The fragmented extracellular matrix components may comprise, at least in part, defibrated extracellular matrix components. Alternatively, the fragmented extracellular matrix components may be composed only of the extracellular matrix components that have been defibrated. That is, the fragmented extracellular matrix components may be defibrated extracellular matrix components. The fibrillated extracellular matrix component preferably comprises a fibrillated collagen component (fibrillated collagen component). The fibrillating collagen component preferably maintains the triple helix structure of the collagen source. By dispersing the fragmented collagen component in an aqueous medium, the fragmented collagen component can be easily brought into contact with cells in the aqueous medium, and the formation of a three-dimensional tissue body can be promoted.

Examples of the shape of the fragmented extracellular matrix component include a fibrous shape. The fibrous shape is a shape formed by a filamentous extracellular matrix component or a shape formed by intermolecular crosslinking of a filamentous extracellular matrix component. At least a portion of the fragmented extracellular matrix components may be fibrous. The fibrous extracellular matrix component includes a filamentous material (fibril) formed by aggregating a plurality of filamentous extracellular matrix molecules, a filamentous material formed by further aggregating fibrils, a substance obtained by fibrillating these filamentous materials, and the like. In the fibrous extracellular matrix component, the RGD sequence is preserved without being destroyed, and can function more effectively as a scaffold material for cell adhesion.

The average length of the fragmented extracellular matrix components may be 100nm or more and 400 μm or less, or may be 100nm or more and 200 μm or less. In one embodiment, the average length of the fragmented extracellular matrix components may be 5 μm or more and 400 μm or less, 10 μm or more and 400 μm or less, 22 μm or more and 400 μm or less, or 100 μm or more and 400 μm or less, from the viewpoint of easy formation of thick tissues. In another embodiment, the average length of the fragmented extracellular matrix components may be 100 μm or less, may be 50 μm or less, may be 30 μm or less, may be 15 μm or less, may be 10 μm or less, may be 1 μm or less, and may be 100nm or more from the viewpoint of easy stabilization of tissue formation and more excellent redispersibility. The average length of the majority of the fragmented extracellular matrix components in the whole of the fragmented extracellular matrix components is preferably within the above numerical range. Specifically, it is preferable that the average length of 95% of the fragmented extracellular matrix components in the entirety of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average length within the above range, and more preferably a defibrinated collagen component having an average length within the above range.

The average diameter of the fragmented extracellular matrix components may be from 50nm to 30 μm, may be from 4 μm to 30 μm, and may be from 5 μm to 30 μm. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average diameter within the above-described range, and more preferably a defibrinated collagen component having an average diameter within the above-described range.

The ranges of the average length and the average diameter are optimized from the viewpoint of tissue formation, and therefore, it is desirable that the range of the average length or the average diameter is limited to the range of the average length or the average diameter at the stage of tissue formation by suspending the fragmented extracellular matrix components in an aqueous medium again after the drying step described later.

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

At least a portion of the fragmented extracellular matrix components may be cross-linked either intermolecularly or intramolecularly. The fragmented extracellular matrix components may be cross-linked intramolecularly, or intermolecularly, which make up the fragmented extracellular matrix components.

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

In the case where the extracellular matrix component contains a collagen component, crosslinking may be formed between collagen molecules (triple helix structure) or between collagen fibrils formed of collagen molecules. The crosslinking may be crosslinking by heat (thermal crosslinking). Thermal crosslinking can be carried out, for example, by heat treatment under reduced pressure using a vacuum pump. In the case of thermal crosslinking of collagen components, extracellular matrix components can be crosslinked by the amino groups of the collagen molecule forming peptide bonds (-NH-CO) with the carboxyl groups of the same or other collagen molecules.

Extracellular matrix components may also be crosslinked by the use of crosslinking agents. The crosslinking agent may be, for example, a crosslinking agent capable of crosslinking a carboxyl group and an amino group, or a crosslinking agent capable of crosslinking amino groups with each other. As the crosslinking agent, for example, aldehydes, carbodiimides, epoxides and imidazole crosslinking agents are preferable from the viewpoint of economy, safety and handling, and specific examples thereof include water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide sulfonate.

The crosslinking degree can be determined appropriately according to the kind of the extracellular matrix component, the means for crosslinking, and the like. The degree of crosslinking 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 crosslinking degree is within the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after dry storage is good.

When the amino group in the extracellular matrix component is used for crosslinking, the degree of crosslinking can be determined by the TNBS method described in non-patent document 2 and the like. The degree of crosslinking by the TNBS method is preferably within the above range. The degree of crosslinking by the TNBS method is the proportion of amino groups for crosslinking among amino groups of the extracellular matrix.

The degree of crosslinking can also be calculated by quantifying the carboxyl groups. For example, in the case of an extracellular matrix component insoluble in water, the amount can be determined by the TBO (toluidine blue O) method. The degree of crosslinking by the TBO method may be in the above-mentioned range.

The content of the fragmented extracellular matrix components (fragmented extracellular matrix components) in the extracellular matrix-containing composition may be 1 mass% or more, 3 mass% or more, 10 mass% or more, 20 mass% or more, 30 mass% or more, 40 mass% or more, 50 mass% or more, 60 mass% or more, 70 mass% or more, 80 mass% or more, 90 mass% or more, 95 mass% or more, or 98 mass% or more, and may be 99 mass% or less, 95 mass% or less, or 90 mass% or less, based on the total amount of the extracellular matrix-containing composition.

The extracellular matrix-containing composition of this embodiment comprises a compound bound or adsorbed to a fragmented extracellular matrix component. The compound may be a biomolecule as long as it is capable of binding to or adsorbing to the fragmented extracellular matrix components contained in the extracellular matrix-containing composition. In this specification, the ability to bind or adsorb is synonymous with the ability to coat a defibrinated extracellular matrix component with a compound. In the case of applying the defibrated extracellular matrix component, the whole surface may be applied, or a part may be applied. The compound may be appropriately selected depending on the kind of the extracellular matrix component to be fragmented. The compound may be, for example, an extracellular matrix component comprising an extracellular matrix molecule that is the same species or different species as the fragmented extracellular matrix component. Specific examples of the compound include, but are not limited to, chondroitin sulfate, heparan sulfate, fibronectin, heparin, proteoglycan, hyaluronic acid, heparan sulfate proteoglycan, chondroitin sulfate proteoglycan, laminin, entactin, cell adhesin, elastin, and fibrin. For example, the compound may be at least 1 selected from heparan sulfate, chondroitin sulfate, and fibronectin. In the case where the fragmented extracellular matrix component is a fragmented collagen component, the compound may be chondroitin sulfate, heparan sulfate, heparin or fibronectin. The fragmented extracellular matrix component to which the compound is bound or adsorbed has a function that the fragmented extracellular matrix component alone does not have, or may enhance the function of the fragmented extracellular matrix component alone. For example, in the case of chondroitin sulfate, the adhesion of cells to each other is increased. In the case of heparin, cell growth becomes easy. In the case of fibronectin, the adhesion of the fragmented extracellular matrix components to the cells is improved.

The content of the compound may be 0.5 parts by mass or more and 10 parts by mass or less, 1.0 parts by mass or more and 8.0 parts by mass or less, and 1.5 parts by mass or more and 4.0 parts by mass or less, with respect to 100 parts by mass of the fragmented extracellular matrix component. The content of the compound can be calculated based on, for example, the adsorption rate described in examples described later.

The extracellular matrix-containing composition may be composed of only the fragmented extracellular matrix component and the above-mentioned compound, or may contain a component (other component) other than the fragmented extracellular matrix component and the above-mentioned compound.

The extracellular matrix-containing composition may be in a solid or powder form, from the viewpoint of easy weighing. The extracellular matrix-containing composition may not contain moisture. The water in the extracellular matrix-containing composition can be removed, for example, by freeze-drying. The absence of moisture does not mean that any water molecule is not included, but means that moisture is not included to the extent that can be achieved by the drying method such as freeze drying in a common sense.

The extracellular matrix-containing composition of one embodiment may be dispersed in an aqueous medium. The term "aqueous medium" refers to a liquid containing water as an essential component. The aqueous medium is not particularly limited as long as the extracellular matrix component can be stably present. Examples of the aqueous medium include, but are not limited to, a physiological saline such as Phosphate Buffered Saline (PBS), a liquid medium such as Dulbecco's Modified Eagle Medium (DMEM), and a vascular endothelial cell-specific medium (EGM 2).

The dispersibility in an aqueous medium can be determined, for example, by the following method. That is, when 50mg of the extracellular matrix-containing composition was added to 5mL of ultrapure water and suspended, it was judged that the composition could be dispersed in an aqueous medium when the extracellular matrix-containing composition was dispersed in ultrapure water (when aggregation or the like did not occur). The temperature for dispersing the extracellular matrix-containing composition in ultrapure water may be a temperature not higher than the culture temperature (for example, 37 ℃) or may be room temperature. The dispersed state means a state in which aggregation, sedimentation, or the like does not occur visually. The dispersibility may be determined by, for example, measuring absorbance.

The pH of the aqueous medium is preferably in a range that does not adversely affect the growth of cells or the formation of cell aggregates. The pH of the aqueous medium may be, for example, 7.0 or more and 8.0 or less from the viewpoint of reducing the load on cells when the cells are put into the medium. Specifically, the pH of the aqueous medium may be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The aqueous medium preferably has a buffering capacity in the above-mentioned pH range, and more preferably is a liquid medium. The liquid medium is not particularly limited, and an appropriate medium can be selected depending on the type of cells to be cultured. Examples of the medium include Eagle's MEM medium, DMEM, Modified Eagle Medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be a serum-supplemented medium or a serum-free medium. The liquid medium may be a mixed medium obtained by mixing two or more kinds of media.

< method for producing extracellular matrix-containing composition >

The method for producing an extracellular matrix-containing composition according to the present embodiment includes a step (contact step) of contacting a fragmented extracellular matrix component with a compound that can be bonded to or adsorbed to the fragmented extracellular matrix component. Examples of the contacting step include, but are not limited to, mixing an aqueous medium containing the fragmented extracellular matrix components with an aqueous medium containing a compound, and adding the compound to the aqueous medium containing the fragmented extracellular matrix components. The contacting step may include a step of incubating the fragmented extracellular matrix component and the compound for a certain period of time after contacting them.

The fragmented extracellular matrix components can be obtained by the above-described method. The fragmented extracellular matrix components may be obtained by fragmenting extracellular matrix components in an aqueous medium. That is, the production method of the present embodiment may include a step (fragmentation step) of fragmenting the extracellular matrix component in an aqueous medium before the contacting step. The aqueous medium may be the same as the aqueous medium described above. The fragmentation step may be a step of defibrating the extracellular matrix components in an aqueous medium before the contacting step.

The fragmented extracellular matrix component may be the one exemplified above, and may contain a fragmented collagen component or a defibrated collagen component. The compounds that can be bound or adsorbed to the fragmented extracellular matrix components may use the compounds described above.

The production method of the present embodiment may include a step of heating the extracellular matrix component to crosslink at least a part of the extracellular matrix component before the fragmentation step, or may include a step of heating the extracellular matrix component to crosslink at least a part of the extracellular matrix component after the fragmentation step and before the contact step.

In the step of crosslinking, the temperature (heating temperature) and time (heating time) for heating the extracellular matrix component can be determined as appropriate. The heating temperature may be, for example, 100 ℃ or higher and 200 ℃ or lower. The heating temperature may be, for example, 100 ℃, 110 ℃, 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, 170 ℃, 180 ℃, 190 ℃, 200 ℃ or the like. The heating time (the time for holding at the heating temperature) can be set as appropriate depending on the heating temperature. The heating time may be 6 hours or more and 72 hours or less, and more preferably 24 hours or more and 48 hours or less, for example, when heating is performed at 100 to 200 ℃. In the step of crosslinking, heating may be performed in the absence of a solvent or under reduced pressure.

The production method of the present embodiment may include a drying step of drying the fragmented extracellular matrix components after the fragmentation step.

In the drying process, the fragmented extracellular matrix components are dried. The drying may be carried out, for example, by freeze drying. The aqueous medium is removed from the liquid comprising the fragmented extracellular matrix components and the aqueous medium by performing a drying process after the fragmentation process. The removal of the aqueous medium does not mean that any moisture is not attached to the fragmented extracellular matrix components, but means that moisture is not attached to a degree that can be achieved by the conventional drying method as described above, which is common knowledge.

The extracellular matrix-containing composition may be suitably used as a scaffold material for forming three-dimensional tissue bodies. Thus, the extracellular matrix-containing composition is suitable for use in three-dimensional tissue formation applications.

< three-dimensional tissue-forming agent >

The extracellular matrix-containing composition is suitable as a scaffold material or the like for forming a three-dimensional tissue body, and therefore in one embodiment of the present invention, there is provided a three-dimensional tissue body-forming agent comprising the extracellular matrix-containing composition described above.

The three-dimensional tissue forming agent of the present embodiment includes the defibrated extracellular matrix component described above, and thus can form a thicker three-dimensional tissue.

The three-dimensional structure forming agent may be in a powder state during storage, and is preferably in a dispersion liquid state dispersed in an aqueous medium at the stage of forming a three-dimensional structure body.

< three-dimensional tissue body >

The three-dimensional tissue body of the present embodiment includes the extracellular matrix-containing composition and the cells. At least a portion of the cells can be contacted with a defibrinated extracellular matrix component of an extracellular matrix-containing composition. As one way of contacting, adhesion may be performed. The "three-dimensional tissue body" refers to an aggregate of cells in which cells are three-dimensionally arranged via an extracellular matrix component, and refers to an aggregate artificially produced by cell culture. The shape of the three-dimensional structure is not particularly limited, and examples thereof include a sheet shape, a spherical shape, an elliptical shape, and a rectangular parallelepiped shape. Here, the living tissue includes blood vessels, sweat glands, lymph vessels, sebaceous glands, and the like, and is more complex than a three-dimensional tissue. Therefore, the three-dimensional tissue body can be easily distinguished from the living tissue.

The cell is not particularly limited, and may be a cell derived from an animal such as a human, monkey, dog, cat, rabbit, pig, cow, mouse, or rat. The origin of the cells is not particularly limited, and may be somatic cells derived from bone, muscle, viscera, nerve, brain, bone, skin, blood, etc., or germ cells. The cell may be an induced pluripotent stem cell (iPS cell), an embryonic stem cell (ES cell), or a cultured cell such as a primary cultured cell, a subculture cell, or a cell line cell. Specifically, examples of the cells include nerve cells, dendritic cells, immune cells, vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVECs)), perivascular endothelial cells, lymphatic endothelial cells, fibroblasts, colorectal cancer cells (e.g., human colorectal cancer cells (HT29)), cancer cells such as liver cancer cells, epithelial cells (e.g., human gingival epithelial cells), keratinocytes, cardiac myocytes (e.g., human iPS cell-derived cardiac myocytes (iPS-CM)), hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells (e.g., Aorta-SMC), and the like, but are not limited thereto, and the cells may include one or more cells selected from vascular endothelial cells, cancer cells, cardiac myocytes, smooth muscle cells, and epithelial cells, a plurality of cells may be used in combination.

The cell is preferably an extracellular matrix-secreting cell that secretes an extracellular matrix molecule. Examples of the extracellular matrix-secreting cells include collagen-secreting cells that secrete collagen such as fibrous collagen. Examples of the collagen-secreting cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, and fibroblasts are preferred. Preferred fibroblasts include human skin-derived fibroblasts (NHDF), human cardiac fibroblasts (NHCF), and Human Gingival Fibroblasts (HGF).

In the case where the three-dimensional tissue body contains extracellular matrix-secreting cells as cells, the three-dimensional tissue body may contain endogenous extracellular matrix. "endogenous extracellular matrix" refers to the extracellular matrix produced by extracellular matrix-producing cells that make up a three-dimensional tissue body.

In the case where the three-dimensional tissue body contains collagen-secreting cells as cells, the three-dimensional tissue body may contain endogenous collagen. "endogenous collagen" refers to collagen produced by collagen-producing cells that make up a three-dimensional tissue body. The endogenous collagen may be fibrillar collagen or non-fibrillar collagen.

In the case where the three-dimensional tissue body contains extracellular matrix-secreting cells as cells, the three-dimensional tissue body may contain: a cell comprising an extracellular matrix-secreting cell, an extracellular matrix-containing composition, and an endogenous extracellular matrix component. In this case, at least a portion of the cells comprising the extracellular matrix-secreting cells can be contacted with the fragmented extracellular matrix components and/or endogenous extracellular matrix components. Conventional three-dimensional tissue bodies have a low extracellular matrix (e.g., collagen) concentration and a high cell density. Therefore, there are problems as follows: the three-dimensional tissue body is easily decomposed by contraction due to traction of the cells during or after the culture, or by an enzyme produced by the cells during or after the culture. In the three-dimensional tissue body according to one embodiment, the concentration of extracellular matrix (collagen or the like) is higher than that of a conventional tissue body, and the tissue body is less likely to cause contraction and is stable.

The three-dimensional tissue body may include extracellular matrix-secreting cells and cells other than the extracellular matrix-secreting cells as cells. Examples of the cells other than the extracellular matrix-producing cells include vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVECs)), colorectal cancer cells (e.g., human colorectal cancer cells (HT29)), cancer cells such as liver cancer cells, cardiac muscle cells (e.g., human iPS cell-derived cardiac muscle cells (iPS-CM)), epithelial cells (e.g., human gingival epithelial cells), keratinocytes, lymphatic endothelial cells, nerve cells, liver cells, tissue stem cells, embryonic stem cells, artificial pluripotent stem cells, adhesive cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Aorta-SMC)), and the like. The cells constituting the three-dimensional tissue body preferably further include one or more cells selected from the group consisting of vascular endothelial cells, cancer cells, cardiac muscle cells, smooth muscle cells, and epithelial cells.

The total content of the fragmented extracellular matrix component and the compound in the three-dimensional tissue body may be 0.01 mass% or more and 90 mass% or less, may be 10 mass% or more and 80 mass% or less, may be 10 mass% or more and 70 mass% or less, may be 10 mass% or more and 60 mass% or less, may be 1 mass% or more and 50 mass% or less, may be 10 mass% or more and 30 mass% or less, and may be 20 to 30 mass% based on the total mass of the fragmented extracellular matrix component, the compound, and the cells. The total content of fragmented extracellular matrix components and the above-mentioned compounds can be determined, for example, by conventional MS imaging.

When the three-dimensional tissue body contains collagen, the collagen content in the three-dimensional tissue body may be 0.01 to 90% by mass, preferably 0.33 to 90% by mass, preferably 5 to 90% by mass, preferably 10 to 80% by mass, preferably 10 to 70% by mass, preferably 10 to 60% by mass, preferably 1 to 50% by mass, preferably 10 to 50% by mass, more preferably 10 to 30% by mass, and still more preferably 20 to 30% by mass, based on the total mass of the fragmented extracellular matrix component, the compound, and the cells.

Here, the "collagen in the three-dimensional tissue body" refers to collagen constituting the three-dimensional tissue body, and may be endogenous collagen or collagen derived from a fragmented collagen component (exogenous collagen). That is, in the case where the three-dimensional tissue body includes an endogenous collagen component and a fragmented collagen component, the collagen content rate constituting the three-dimensional tissue body refers to the total concentration of the endogenous collagen component and the fragmented collagen component. The collagen content can be calculated from the volume of the obtained three-dimensional tissue body and the mass of the decellularized three-dimensional tissue body.

In addition, as a method for quantifying the collagen amount in the three-dimensional tissue body, for example, the following method for quantifying hydroxyproline is exemplified. A sample is prepared by mixing hydrochloric acid (HCl) with a solution in which a three-dimensional tissue is dissolved, incubating the mixture at a high temperature for a predetermined time, returning the temperature to room temperature, and diluting the supernatant after centrifugation to a predetermined concentration. The hydroxyproline standard solution was treated in the same manner as the sample, and then diluted stepwise to prepare a standard solution. The sample and the standard solution were subjected to predetermined treatments with a hydroxyproline assay buffer and a detection reagent, respectively, and the absorbance at 570nm was measured. The collagen amount was calculated by comparing the absorbance of the sample with a standard solution. The three-dimensional tissue may be directly suspended in high-concentration hydrochloric acid, and the dissolved solution may be centrifuged to collect the supernatant and used for collagen quantification. The dissolved three-dimensional tissue may be directly collected from the culture medium, or may be subjected to a drying treatment after collection to dissolve the tissue in a state where liquid components are removed. However, when a collagen is quantified by dissolving a three-dimensional tissue body collected directly from a culture solution, the measurement value of the weight of the three-dimensional tissue body is expected to vary due to the influence of the components of the culture medium absorbed by the three-dimensional tissue body and the residual culture medium due to the problem of the experimental technique, and therefore, it is preferable to use the weight after drying as a reference from the viewpoint of stably measuring the weight of the tissue body and the amount of collagen per unit weight.

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

(preparation of sample)

The total amount of the three-dimensional tissue body subjected to the freeze-drying treatment was mixed with 6mol/l HCl, incubated at 95 ℃ for 20 hours or more with a heating block, and then returned to room temperature. After centrifugation at 13000g for 10 minutes, the supernatant of the sample solution was recovered. In the measurement described later, the sample was prepared by diluting appropriately with 6mol/L HCl so that the result falls within the range of the standard curve, and then diluting 200. mu.L with 100. mu.L of ultrapure water. 35 μ L of sample was used.

(preparation of Standard solution)

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

(analysis)

mu.L of the standard solution and sample were added to a plate (QuickZyme Total gel Assay kit attached, QuickZyme Biosciences, Inc.) separately. Add 75. mu.L of assay buffer (attached to the kit above) to each well. The plate was closed with a sealer and incubated at room temperature while shaking for 20 minutes. The seal was peeled off, and 75 μ L of a detection reagent (reagent a: B: 30 μ L: 45 μ L, attached to the above-described kit) was added to each well. The plate was closed with a sealer, the solution was mixed by shaking, and incubated at 60 ℃ for 60 minutes. After cooling sufficiently on ice, the seal was peeled off, and the absorbance at 570nm was measured. The collagen amount was calculated by comparing the absorbance of the sample with a standard solution.

In addition, the collagen occupied in the three-dimensional tissue body can be determined by the area ratio or the volume ratio thereof. The term "determined by the area ratio or the volume ratio" means that, for example, the collagen in the three-dimensional tissue body is in a state distinguishable from other tissue constituents by a known staining method (for example, immunostaining using an anti-collagen antibody or masson trichrome staining) or the like, and then the ratio of the collagen existing region in the entire three-dimensional tissue body is calculated by using visual observation, various microscopes, image analysis software, or the like. When the area ratio is determined, the area ratio is not limited to what cross section or surface the three-dimensional tissue body has, and may be determined by a cross section passing through a substantially central portion of the three-dimensional tissue body, for example, when the three-dimensional tissue body is a spheroid or the like.

For example, when collagen in a three-dimensional tissue is determined by the area ratio, the ratio of the area is 0.01 to 99%, preferably 1 to 99%, preferably 5 to 90%, preferably 7 to 90%, preferably 20 to 90%, and more preferably 50 to 90% based on the area of the entire three-dimensional tissue. The "collagen in the three-dimensional tissue body" is as described above. When the three-dimensional tissue body contains exogenous collagen, the ratio of the area of collagen constituting the three-dimensional tissue body is the ratio of the area of the combination of endogenous collagen and exogenous collagen. The ratio of the collagen area can be calculated, for example, by staining the obtained three-dimensional tissue body with masson trichrome and calculating the ratio of the blue-stained collagen area to the entire area of a cross section passing through the substantially central portion of the three-dimensional tissue body.

The three-dimensional tissue body preferably has a residual ratio of 70% or more, more preferably 80% or more, and even more preferably 90% or more after being subjected to trypsin treatment under conditions of a trypsin concentration of 0.25%, a temperature of 37 ℃, a pH of 7.4, and a reaction time of 15 minutes. Such a three-dimensional tissue body is not easily decomposed by an enzyme during or after culture, and is stable. The residual ratio can be calculated from the mass of the three-dimensional tissue before and after the trypsin treatment, for example.

The three-dimensional structure may have a residual ratio of 70% or more, more preferably 80% or more, and still more preferably 90% or more after the collagenase treatment under the conditions of a collagenase concentration of 0.25%, a temperature of 37 ℃, a pH of 7.4, and a reaction time of 15 minutes. Such a three-dimensional tissue body is not easily decomposed by an enzyme during or after culture, and is stable.

The thickness of the three-dimensional structure is preferably 10 μm or more, more preferably 100 μm or more, and further preferably 1000 μm or more. Such a three-dimensional tissue body has a structure closer to a biological tissue, and is suitable as a substitute for an experimental animal and a transplant material. The upper limit of the thickness of the three-dimensional structure is not particularly limited, and may be, for example, 10mm or less, 3mm or less, 2mm or less, 1.5mm or less, or 1mm or less.

Here, the "thickness of the three-dimensional structure" refers to a distance between both ends in a direction perpendicular to the main surface when the three-dimensional structure is in a sheet shape or a rectangular parallelepiped shape. When the main surface has irregularities, the thickness is the distance of the thinnest part of the main surface.

When the three-dimensional structure is spherical, the thickness refers to the diameter thereof. When the three-dimensional structure is an ellipse, the thickness is the minor axis thereof. When the three-dimensional structure body has a substantially spherical shape or a substantially elliptical shape and has irregularities on the surface, the thickness is the shortest distance among the distances between 2 points at which a straight line passing through the center of gravity of the three-dimensional structure body intersects the surface.

< method for producing three-dimensional structure body >

The method for manufacturing a three-dimensional structure according to the present embodiment includes: a first step of contacting the extracellular matrix-containing composition with cells in an aqueous medium, and a second step of culturing the cells contacted with the extracellular matrix-containing composition. In the method for producing a three-dimensional tissue body according to the present embodiment, it is important that the fragmented extracellular matrix component is brought into contact with a compound before the fragmented extracellular matrix component is brought into contact with cells (i.e., the first step), and the compound is bonded to or adsorbed to the fragmented extracellular matrix component. Thus, a function useful for fragmenting an extracellular matrix component can be imparted or enhanced as compared with the case where cells are cultured by adding only a compound to a cell culture solution. In addition, since the compound is bonded or adsorbed to the fragmented extracellular matrix component, there is also an effect that the amount of the compound used can be suppressed.

In the method for producing a three-dimensional tissue body, the cells are preferably cells including collagen-producing cells. By using cells containing collagen-secreting cells, a three-dimensional tissue body in which cells are distributed more stably and uniformly can be obtained. The details of the mechanism for obtaining such a three-dimensional tissue are not clear, but are presumed as follows.

In the conventional method for producing a three-dimensional tissue using a scaffold, since target cells are injected into a scaffold prepared in advance, it is difficult to uniformly distribute the cells in the scaffold. In the case where the cells are cells comprising extracellular matrix-producing cells (collagen-producing cells, etc.), first, the cells are adhered to the extracellular matrix-containing composition. Thereafter, the cells themselves produce proteins (e.g., collagens such as fibrillar collagen) that constitute extracellular matrix components. The produced protein adheres to the extracellular matrix-containing composition, and thereby functions as a crosslinking agent between the extracellular matrix-containing compositions, and under an environment in which cells are uniformly present, the structuring of proteins and the like constituting extracellular matrix components progresses. As a result, a three-dimensional tissue body in which cells are distributed more stably and uniformly can be obtained. However, the above presumption is not intended to limit the present invention.

In the production methods described in patent documents 1 to 3, the number of steps for producing the three-dimensional structure is large, and a working time of about 1 hour is required. According to the manufacturing method of the present embodiment, the three-dimensional structure can be manufactured in a short work time. Further, according to the manufacturing method of the present embodiment, a three-dimensional structure can be easily manufactured. In the production method described in patent document 2, in order to produce a three-dimensional tissue body having a thickness of about 1mm, it is necessary that the number of cells is at least 10⁶cells. According to the production method of the present embodiment, a large three-dimensional tissue body having a thickness of 1mm or more can be produced with a small number of cells.

In the first step, the extracellular matrix-containing composition is brought into contact with cells in an aqueous medium. The method of contacting the extracellular matrix-containing composition with cells in an aqueous medium is not particularly limited. For example, there may be mentioned a method of adding an extracellular matrix-containing composition to a culture solution containing cells, a method of adding an aqueous medium and cells to an extracellular matrix-containing composition, or a method of adding an extracellular matrix-containing composition and cells separately to a previously prepared aqueous medium.

In the first step, cells including collagen-producing cells and cells other than the collagen-producing cells may be used. The cells described above can be used as the collagen-producing cells and the cells other than the collagen-producing cells, respectively. By using collagen-producing cells together with cells other than the collagen-producing cells to produce a three-dimensional tissue body, various model tissues can be produced. For example, when NHCF and HUVEC are used, a three-dimensional tissue body having capillaries therein can be obtained. When NHCF and colon cancer cells are used, a model tissue of colon cancer can be obtained. When NHCF and iPS-CM were used, a model tissue of the myocardium showing palpitations was obtained.

The concentration of the extracellular matrix-containing composition in the first step may be appropriately determined depending on the shape and thickness of the target three-dimensional tissue body, the size of the culture device, and the like. For example, the concentration of the extracellular matrix-containing composition in the aqueous medium in the first step may be 0.1 to 90% by mass, or 1 to 30% by mass.

The amount of the extracellular matrix-containing composition in the first step was 1X 10 times the amount of the composition⁵The cells may be 0.1 to 100mg, or 1 to 50 mg.

In the first step, the mass ratio of the extracellular matrix-containing composition to the cells (extracellular matrix-containing composition/cells) is preferably 1/1 to 1000/1, more preferably 9/1 to 900/1, and still more preferably 10/1 to 500/1.

When the collagen-producing cells are used together with other cells, the ratio of the number of cells of the collagen-producing cells to the ratio of the number of other cells in the first step (the ratio of the collagen-producing cells to the other cells in the first step) may be 9/1 to 99/1, 50/50 to 80/20, 20/80 to 50/50, or 10/90 to 50/50.

The method may further comprise a step of sedimenting the extracellular matrix-containing composition in the aqueous medium together with the cells after the first step and before the second step. By performing such a step, the extracellular matrix-containing composition and the cells in the three-dimensional tissue body are more uniformly distributed. Specific methods are not particularly limited, and examples thereof include a method of centrifuging a culture solution containing an extracellular matrix-containing composition and cells.

The first step may be performed after the cell layer is formed in the aqueous medium. That is, the first step may be performed by forming a cell layer in an aqueous medium and then contacting the extracellular matrix-containing composition. By forming the cell layer before contacting the extracellular matrix-containing composition, a three-dimensional tissue body having a high cell density in the lower layer can be produced. In addition, by forming the layer containing cells that produce collagen before contacting the extracellular matrix-containing composition, a three-dimensional tissue body with a high cell density in the lower layer of cells that produce collagen can be produced. Depending on the type of cells used (for example, aortic smooth muscle cells), a tissue closer to a living body can be prepared by this method.

After the second step, a step of culturing the cells by bringing the cells into contact may be further included as a third step. The cells may be of the same type as the cells used in the first step, or of different types. For example, in the case where the cells used in the first step include cells other than collagen-producing cells, the cells used in the third step may include collagen-producing cells. For example, when the cells used in the first step include collagen-producing cells, the cells used in the third step may include cells other than collagen-producing cells. Both the cells used in the first step and the cells used in the third step may include collagen-producing cells, and both the cells used in the first step and the cells used in the third step may include cells other than collagen-producing cells. By the third step, a three-dimensional tissue body having a double-layer structure can be produced. For example, when aortic smooth muscle cells and vascular endothelial cells are used, or when human skin-derived fibroblasts and human epidermal keratinocytes are used, a tissue closer to a living body can be prepared by this method. In addition, for example, in the case of using human gingival fibroblasts and gingival epithelial cells, a three-dimensional tissue body having a double-layer structure free from tissue contraction and tissue rupture can be produced by this method.

The method for culturing the cells contacted with the extracellular matrix-containing composition is not particularly limited, and may be carried out by an appropriate culturing method depending on the type of the cells to be cultured. For example, the culture temperature may be 20 to 40 ℃ or 30 to 37 ℃. The pH of the culture medium may be 6 to 8 or 7.2 to 7.4. The culture time may be 1 day to 2 weeks, or 1 week to 2 weeks.

The medium is not particularly limited, and an appropriate medium may be selected depending on the kind of cells to be cultured. Examples of the medium include Eagle's MEM medium, DMEM, Modified Eagle Medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be a serum-supplemented medium or a serum-free medium. The liquid medium may be a mixed medium obtained by mixing two or more kinds of media.

The cell density in the medium in the second step can be determined as appropriate depending on the shape and thickness of the target three-dimensional tissue body, the size of the culture container, and the like. For example, the cell density in the medium in the second step may be 1 to 10⁸cells/ml, also may be 10³～10⁷cells/ml. The cell density in the medium in the second step may be the same as the cell density in the aqueous medium in the first step.

The shrinkage rate of the three-dimensional tissue body during culture is preferably 20% or less, more preferably 15% or less, and still more preferably 10% or less. The shrinkage can be calculated by the following formula, for example. Wherein L1 represents the length of the longest portion of the three-dimensional tissue body on day 1 after culture, and L3 represents the length of the corresponding portion in the three-dimensional tissue body on day 3 after culture.

Shrinkage (%) { (L1-L3)/L1} × 100

By the above-mentioned production method, for example, a three-dimensional tissue body including cells and extracellular matrix components can be produced, wherein the collagen content is 10 to 90% by mass based on the three-dimensional tissue body.

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

< test example 1: preparation of defibrated collagen component

A lyophilized body of collagen type I derived from pig skin manufactured by Nippon Ham Co., Ltd was suspended in ultrapure water so that the collagen content became 1 mass%. The suspension was homogenized using an ultrasonic homogenizer by repeating 10 cycles for 20 seconds at 4 ℃. The obtained liquid was filtered through a filter having a pore size of 35 μm to obtain a defibrated collagen component (sCMF).

The mean length (length) of the sccmf was 14.8 ± 8.2 μm (N ═ 20).

< test example 2: adsorption of defibrated collagen component (sCMF) to biomolecules >

The following compounds 1 to 4 were prepared.

Compound 1: sodium hyaluronate labeled with aminofluorescein (PG Research, FAHA-H1)

Compound 2: aminofluorescein labeled heparan sulfate sodium (PG Research, FAHS-P1)

Compound 3: aminofluorescein labeled chondroitin sulfate A sodium (PG Research, FACS-A1)

Compound 4: rhodamine-labeled fibronectin (Cytoskelton, inc., Rhodamine fibronectin)

Compounds 1 to 4 were diluted with Phosphate Buffered Saline (PBS) to prepare a solution containing any of compounds 1 to 4 at a concentration of 0.04 mg/ml.

To 1mg of sCMF was added 1mL of the prepared solution to obtain a mixed solution. The mixture was stirred at 20rpm at room temperature (15 to 25 ℃ C.) for 60 minutes. After completion of the stirring, the mixture was centrifuged, and the supernatant was removed and washed with PBS to prepare a sample for testing. Fig. 1 shows the observation results of the test solutions prepared using any of compounds 1 to 4.

Phase difference (Ph) observation and fluorescence observation confirmed that the compound was bound or adsorbed to the sccmf in the test samples prepared using the aminofluorescein-labeled heparan sulfate sodium, the aminofluorescein-labeled chondroitin sulfate a sodium, and the rhodamine-labeled fibronectin, respectively. On the other hand, in the test sample prepared by using the aminofluorescein-labeled sodium hyaluronate, it was not confirmed that the compound was bonded or adsorbed to the sccmf.

< test example 3: evaluation of adsorption Rate of biomolecule

Solutions (referred to as solutions 1 to 3, respectively) were prepared by diluting compound 1(HA), compound 2(CS), and compound 3(HS) with PBS so that the concentrations thereof became 0.04 mg/ml.

Solutions 1 to 3 were diluted with PBS to prepare solutions for standard curve preparation so that the concentrations of the compounds 1 to 3 became 0.1mg/ml, 0.05mg/ml, 0.025mg/ml, or 0.0125 mg/ml. The fluorescence intensity of the solution for preparing the calibration curve was measured using a spectrofluorometer (Japanese Spectroscopy, FP-8500) to prepare a calibration curve.

1mg of sCMF was added to a 1.5ml sample tube (WATSON, 131. sub. 7155C), and any of 1 to 3 ml of the solutions was added, followed by stirring at room temperature at 20rpm for 1 hour using ROTATOR (TAITEC, RT-50). The resulting mixture was centrifuged at 3500rpm for 5 minutes by a centrifugal separator (eppendorf, minispin), and the fluorescence intensity of 200. mu.L of the supernatant was measured by a spectrofluorometer. The obtained results were compared with a standard curve, and the concentration of the fluorescent reagent in the supernatant was calculated therefrom. When this value is X, the adsorption rate is calculated by the formula (0.04-X)/0.04X 100. The results of the adsorption rate measurement are shown in table 1.

TABLE 1

< test example 4: evaluation of adsorption stability

1mg of sCMF was added to a 1.5ml sample tube (WATSON, 131. sub. 7155C) and 1ml of a solution containing Compound 2 was added (preparation of 8 tubes). The mixture was stirred at 20rpm for 1 hour at room temperature using ROTATOR (TAITEC, RT-50). The supernatant was centrifuged at 3500rpm for 5 minutes using a centrifugal separator (eppendorf, minispin), and the fluorescence intensity of 200. mu.l of each supernatant was measured using a spectrofluorometer (Japanese Spectroscopy, FP-8500). The obtained fluorescence intensity was compared with a standard curve, and the concentration of the fluorescent reagent in the supernatant was calculated. This is denoted as X. The supernatants were aspirated and 1ml PBS was added. After 5, 20, 40, 60, 120, 300, 1440 or 5760 minutes, the supernatants were centrifuged at 3500rpm for 5 minutes by a centrifuge, and the fluorescence intensity of 200. mu.l of each supernatant was measured by a spectrofluorometer. The obtained fluorescence intensity was compared with a standard curve, and the concentration of the fluorescent reagent in the supernatant was calculated. This is denoted as Y. According to the adsorption stability index: the adsorption stability was evaluated by the formula (X-Y)/X.times.100. Fig. 2 is a photomicrograph showing the test results after 5760 minutes.

TABLE 2

Time (minutes)	Index of adsorption stability
		0	100
5	83
		20	79
40	71
		60	77
120	67
		300	71
1440	43
		5760	12

As shown in table 2, it was shown that compound 2 was still bound and/or adsorbed to the sccmf even after 4 days (5760 minutes) had passed.

< test example 5: evaluation of three-dimensional tissue Using sCMF-FN 1 >

5MG of sCMF was added to 5ml of a 0.04% fibronectin (SIGMA, F2006-5MG) solution in 50mM tris-HCl and stirred at 20rpm for 1 hour at room temperature using ROTATOR (TAITEC, RT-50). The agitation is terminated immediately before mixing with the cells. The resulting mixture was centrifuged at 3500rpm for 5 minutes using a centrifugal separator (eppendorf, minispin). The supernatant was aspirated and 300. mu.L of medium was added. The sCMF produced by these operations is referred to as sCMF-FN.

Mixing 1.0X 10⁶NHDF (Lonza, CC-2509) and 5.0X 10 of cells⁵cells' HUVEC (Lonza, C2517A) were mixed into sCMF-FN as described above and seeded in 24-well inserts (costar, 3470-clear).

This was centrifuged at 1100g for 15 minutes to pellet sCMF-FN and cells, and cultured in 1mL of mixed medium (DMEM (Nacalai Tesque, 08489-45) 50%/EBM-2 (Nacalai Tesque, Lonza, CC-3202) 50%) for 1 day. The thus-obtained culture was transferred to a 6-well plate (IWAKI, 3810-S006) and cultured in 12mL of a mixed medium for 6 days. Immunostaining was performed using CD31(Dako, M0823) and Alexa 647(invitrogen, a21235), fluorescence observation was performed using a confocal quantitative Image cytometer cq (yokogawa), and the diameter of the capillary vessels was determined from the MIP images using Image analysis software (Image J, NIH). The diameter of the capillary is not determined automatically, but by manually drawing a line across the diameter, the length is determined. The same procedure was used for the experiment using sCMF in place of sCMF-FN. The observation result of the fabricated three-dimensional tissue is shown in fig. 5. By using the three-dimensional tissue body produced by sCMF-FN, a three-dimensional tissue body having a thicker vascular network formed therein can be obtained as compared with the three-dimensional tissue body produced by using sCMF.

< test example 6: evaluation of three-dimensional tissue Using sCMF-FN 2 >

A three-dimensional structure was produced in the same manner as in test example 5. The immunostained sections of CD31 and toluidine blue-stained sections were prepared by Applied Medical Service, Inc. using the three-dimensional tissue.

The prepared slice image was photographed using a microscope. The long diameter of the blue-stained nuclei in Toluidine Blue (TB) -stained sections was measured with N-20 using Image analysis software (Image J, NIH). In the immunostained section of CD31, the number of figures in which the immunostained portion (brown) of CD31 was circular and the interior was hollow (white) was measured from the whole image. The observation results of the fabricated three-dimensional structure are shown in fig. 6 and 8. Fig. 7 shows the measurement results of the length of the nucleus in the three-dimensional tissue. By using the three-dimensional tissue body produced by the sCMF-FN, a three-dimensional tissue body in which a thicker lumen is formed is obtained as compared with the three-dimensional tissue body produced by using the sCMF. This is an unexpected effect of the method of mixing fibronectin contributing to cell-to-cell adhesion, and is an unexpected effect exhibited by fibronectin bonding or adsorption to a defibrotized extracellular matrix.