JP2016174538A - Tubular structure, method for producing cell structure, and method for producing tubular structure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell-containing tubular structure that has higher water permeability and higher strength compared with those of the conventional structure consisting of only cells, a method for producing a cell structure, and a method for producing a tubular structure.SOLUTION: A tubular structure comprises a cell structure which comprises a biocompatible polymer block, a fibroblast, a vascular endothelial cell and a smooth muscle cell, where the plurality of polymer blocks are disposed in a gap between the plurality of cells.SELECTED DRAWING: None

Description

  The present invention relates to a tubular structure composed of a cell structure including a biocompatible polymer block and predetermined cells. The present invention further relates to a method for producing a cell structure and a method for producing a tubular structure.

  In vivo, tubular (tubular) structures such as blood vessels, digestive tracts, ureters, and lymphatic vessels function as important tissues. A particularly important tubular structure is a blood vessel, and many artificial blood vessels have been developed due to the need for substitution in disease and surgery. Artificial blood vessels that have been clinically applied include artificial blood vessels using non-biodegradable synthetic polymers such as expanded polyterolafluoroethylene (ePTFE).

  In addition, attempts have been made for a long time to develop a hybrid artificial blood vessel in which an artificial blood vessel made of a synthetic polymer is endothelialized with cells. It has been shown that non-thrombogenicity can be imparted by endothelializing an artificial blood vessel with cells, and an effect of maintaining patency can be expected.

  However, a compliance mismatch has been reported in the anastomosis portion between the vascular graft and the living artery (Non-patent Document 1). Further, it is known that blood flow separation and blood flow stagnation occur at the anastomosis portion between the artificial blood vessel and the living artery, and stress concentration in the anastomosis portion causes the anastomosis blockage (Non-patent Document 2 and Non-Patent Document 2). Reference 3).

  Patent Document 1 describes a support for arranging a cell mass in an arbitrary space, and including a substrate and a filamentous body or a needle-like body for penetrating the cell mass. Patent Document 1 describes that a cell construct is produced by arranging a cell mass in an arbitrary space using the support. Non-Patent Document 4 describes a three-dimensional structure of cardiomyocytes using spheroids composed of cells only and a vascular structure of smooth muscle cells.

  On the other hand, Patent Document 2 describes a cell structure including a polymer block having biocompatibility and a cell, wherein a plurality of the polymer blocks are arranged in a gap between the plurality of cells. Yes. In the cell structure described in Patent Document 2, nutrients can be delivered from the outside to the inside of the cell structure, and the cell structure has a sufficient thickness and cells are uniformly present in the structure. In the example of Patent Document 2, high cell survival activity has been demonstrated using a polymer block made of recombinant gelatin or a natural gelatin material.

Japanese Patent No. 4517125 International Publication WO2011 / 108517

Abbot, W.M .; Megerman, J .; Hasson, J. E .; L'Italien, G .; Warnock, O.F., J. Vasc. Surg. 5 (2): 376-382; 1987 Weston, M.W .; Rhee, K., Tarbell, J.M., J. Biomech. 29 (2): 187-198; 1996 Rhee, K .; Tarbell, J.M., J. Biomech. 27 (3): 329-338; 1994 Shigeki Moriguchi et al., Artificial Organ 41, No. 3, pp. 168-171, 2012

  It is known that an artificial blood vessel made of a synthetic polymer is likely to be clogged due to occlusion and stenosis caused by a thrombus formed on the surface of the material, particularly when the diameter is small. In addition, when the subject is in the growth process of a child or the like, blood vessels that do not self-grow are not suitable. As an attempt to solve the above problem, a hybrid artificial blood vessel in which an artificial blood vessel made of a synthetic polymer material is endothelialized with cells has been reported, but the problem of an anastomotic occlusion at the anastomosis portion between the artificial blood vessel and a living artery is reported. There is. From this point of view, there is a need for a cell-containing bioartificial blood vessel that does not contain a non-bioabsorbable material and can be easily replaced with a living tissue, as well as the need for a size change for a subject such as a child.

  On the other hand, it is known that the wall thickness of a normal artery and aorta is about 1 to 2 mm. When a tubular structure having a wall thickness of 1 to 2 mm is formed with cells alone, various problems arise due to the fact that the liquid cannot diffusely pass through the wall (no water permeability). For example, there is a problem that the inside of the wall cannot be metabolized because there is no water permeability and the liquid cannot be exchanged, and there is a problem that cytokines and hormones produced inside the wall cannot be released to the outside of the wall. Moreover, in the structure produced only with the cells, the strong strength of the structure cannot be obtained, so that the usable parts are limited. In particular, in a tube structure such as a blood vessel or a bile duct, it is known that the strength of the wall is particularly necessary because the liquid flows through the tube at a high flow rate. From this point of view, a structure made only of cells was unsuitable for use in vivo. Furthermore, it is known that a normal tissue is hardly composed of a single cell, and has a function as a tissue by arbitrarily arranging a plurality of types of cells.

  In Patent Document 1 and Non-Patent Document 4, it is described that a three-dimensional structure such as an artificial blood vessel is produced using a spheroid composed only of cells. However, as described above, water permeability is not sufficient and strength is also high. There was a problem that it was not enough. In Patent Document 2, a cell structure containing a biocompatible polymer block and cells is described, but there is no mention of forming a tubular structure such as an artificial blood vessel.

  An object of the present invention is to provide a cell-containing tubular structure having higher water permeability and higher strength than in the case of a structure having only cells. It is another object of the present invention to provide a method for producing a cell structure and a method for producing the tubular structure.

  As a result of intensive studies to solve the above problems, the inventors of the present invention include a biocompatible polymer block, fibroblasts, vascular endothelial cells, and smooth muscle cells, and a plurality of gaps between the cells. A cell structure in which a plurality of the polymer blocks are arranged is passed through the needle-like body of a support provided with a substrate and a needle-like body, and is laminated and cultured, thereby producing the structure only with cells. It was found that a tubular structure having higher water permeability from the inner wall of the tubular structure and capable of improving the strength by further culture can be produced. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A cell structure comprising a biocompatible polymer block, fibroblasts, vascular endothelial cells, and smooth muscle cells, wherein a plurality of the polymer blocks are disposed in a space between the plurality of cells. The tubular structure comprised by this.
(2) The tubular structure according to (1), which is an artificial blood vessel.
(3) The cell structure includes a biocompatible polymer block of 0.0000001 μg or more and 1 μg or less per cell with respect to the total number of fibroblasts, vascular endothelial cells and smooth muscle cells (1) or ( The tubular structure according to 2).
(4) The fibroblast is a dermal fibroblast, the vascular endothelial cell is an umbilical vein vascular endothelial cell, and the smooth muscle cell is a bladder smooth muscle cell, according to any one of (1) to (3) Tubular structure.
(5) The tubular structure according to any one of (1) to (4), wherein the size of the biocompatible polymer block is 10 μm or more and 300 μm or less.

(6) The tubular structure according to any one of (1) to (5), wherein the tubular structure has an inner diameter of 1 mm to less than 6 mm, an outer diameter of 3 mm to 10 mm, and a length of 3 mm to 300 mm.
(7) The tubular structure according to any one of (1) to (6), wherein the biocompatible polymer block is composed of a recombinant peptide or a chemically synthesized peptide.
(8) The tubular structure according to any one of (1) to (7), wherein the recombinant peptide is recombinant gelatin or chemically synthesized gelatin.
(9) The tubular structure according to (8), wherein the recombinant gelatin or the chemically synthesized gelatin is represented by the following formula 1.
Formula 1: A-[(Gly-XY) _n ] _m- B
In formula 1, each of n Xs independently represents any of amino acids, each of N Ys independently represents any of amino acids, m represents an integer of 2 to 10, and n represents 3 to 100 An integer is shown, A represents any amino acid or amino acid sequence, and B represents any amino acid or amino acid sequence.
(10) The above recombinant peptide or chemically synthesized gelatin is
A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;
A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility; or 80% or more of the amino acid sequence described in SEQ ID NO: 1 A peptide having an amino acid sequence having the following sequence identity and having biocompatibility;
The tubular structure according to any one of (7) to (9).
(11) The tubular structure according to any one of (1) to (10), wherein in the biocompatible polymer block, the biocompatible polymer is crosslinked with heat, ultraviolet light, or an enzyme.
(12) The tubular structure according to any one of (1) to (11), wherein the biocompatible polymer block is in the form of granules obtained by pulverizing a porous body of a biocompatible polymer. object.

(13) A method for producing a cell structure comprising a biocompatible polymer block and cells, wherein a plurality of the polymer blocks are disposed in a gap between the plurality of cells, and having an arbitrary shape. There,
The above-mentioned filamentous body or needle-like body of a support comprising a substrate and a filamentous body or needle-like body comprises a biocompatible polymer block and cells, and a plurality of the above-mentioned high Step A in which a plurality of cell structures are disposed, in which molecular blocks are arranged, and the cell structure is arranged so that the arbitrary shape is formed by the plurality of cell structures. A step B of culturing the obtained cell structure and fusing adjacent cell structures together;
A method for producing a cell structure, comprising:
(14) The method for producing a cell structure according to (13), further comprising a step C of pulling out the cell structure formed in an arbitrary shape in the step B from the filamentous body or needle-like body.

(15) A method for producing a tubular structure according to any one of (1) to (12),
The above-mentioned filamentous body or needle-like body of a support comprising a substrate and a filamentous body or needle-like body comprises a biocompatible polymer block and cells, and a plurality of the above-mentioned high Step A1 in which a plurality of cell structures in which molecular blocks are arranged are penetrated, and the cell structure is arranged so that a shape of a tubular structure is formed by the plurality of cell structures, wherein the cells Step B1, comprising fibroblasts, vascular endothelial cells and smooth muscle cells, and culturing the cell structure disposed in step A1 to fuse adjacent cell structures;
A method for manufacturing a tubular structure, comprising:
(16) The method further includes a step C1 of pulling out the cell structure formed in the shape of the tubular structure in the step B1 from the filamentous body or the needle-like body, and a step D1 of culturing the cell structure obtained in the step C1. A method for producing a tubular structure according to 15).

  The tubular structure of the present invention has high wall permeability and high strength. According to the method for producing a cell structure of the present invention, a cell structure having a desired shape can be efficiently produced. According to the method for producing a tubular structure of the present invention, as described above, the tubular structure of the present invention having high wall permeability and high strength can be produced.

FIG. 1 shows a schematic diagram of an example of a tubular structure. FIG. 2 shows a schematic diagram of an example of a support. FIG. 3 shows the temperature profile when freezing the solvent in the examples. FIG. 4 shows an arrangement of a support body having a needle-like body and a cell structure in the embodiment. FIG. 5 shows the results of producing a mosaic cell mass under conditions in which the total number of cells and the amount of CBE3 block were changed. FIG. 6 shows the apparatus used for the water permeability test. FIG. 7 shows the results of testing the water permeability using only the cells produced in Comparative Example 1 using a spheroid-derived tubular structure. FIG. 8 shows the result of water permeability test using the tubular structure derived from the mosaic cell mass produced in Example 5. FIG. 9 shows the relationship between the number of days of culture and the strength of a tubular structure derived from a mosaic cell mass.

Hereinafter, embodiments of the present invention will be described in detail.
[Tubular structure]
The tubular structure of the present invention includes a biocompatible polymer block, fibroblasts, vascular endothelial cells, and smooth muscle cells, and a plurality of the polymer blocks are disposed in a space between the plurality of cells. Cell structure. In addition, the cell structure used in the present invention may be referred to as a mosaic cell mass (a mosaic cell mass) in the present specification.

  The tubular structure of the present invention is a structure having high water permeability and high strength (physical strength), and can be used as, for example, an artificial blood vessel. A cell structure comprising a biocompatible polymer block, fibroblasts, vascular endothelial cells and smooth muscle cells, wherein a plurality of the polymer blocks are disposed in the gaps between the plurality of cells. Having the high performance, i.e. high water permeability and high physical strength, is a completely unexpected and remarkable effect. In Patent Document 1 and Non-Patent Document 4, there is no description about using a polymer block having biocompatibility, and there is no description about a combination of cells such as fibroblasts, vascular endothelial cells and smooth muscle cells. . Patent Document 2 describes a cell structure including a polymer block having bioaffinity and cells, and a plurality of the polymer blocks are arranged in the gaps between the plurality of cells. However, there is no description about forming a tubular structure, there is no description about high water permeability and high physical strength, and there is also a description about a combination of cells such as fibroblasts, vascular endothelial cells and smooth muscle cells. Absent.

(1) Biocompatible polymer block The cell structure used in the present invention includes a biocompatible polymer block. The biocompatible polymer block will be described below.
(1-1) Bioaffinity polymer Bioaffinity means that a significant adverse reaction such as a long-term and chronic inflammatory reaction is not caused upon contact with a living body. The biocompatible polymer used in the present invention is not particularly limited as to whether or not it is degraded in vivo as long as it has affinity for the living body, but is preferably a biodegradable polymer. Specific examples of the non-biodegradable material include polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, stainless steel, titanium, silicone, and MPC (2-methacryloyloxyethyl phosphorylcholine). It is done. Specific examples of biodegradable materials include polypeptides such as recombinant peptides or chemically synthesized peptides (eg, gelatin described below), polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer (PLGA), and hyaluronic acid. , Glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, chitosan and the like. Among the above, a recombinant peptide is particularly preferable. These biocompatible polymers may be devised to enhance cell adhesion. Specifically, “cell adhesion substrate (fibronectin, vitronectin, laminin) and cell adhesion sequence (amino acid single letter notation, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence expressed on the substrate surface) , LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence) coating with peptide, “substrate surface amination, cationization”, or “substrate surface plasma treatment, corona discharge hydrophilicity” Can be used.

The type of polypeptide including recombinant peptide or chemically synthesized peptide is not particularly limited as long as it has biocompatibility. For example, gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, enteractin, thrombo Spongein and retronectin are preferred, and gelatin, collagen and atelocollagen are most preferred. The gelatin for use in the present invention is preferably natural gelatin, recombinant gelatin or chemically synthesized gelatin, and more preferably recombinant gelatin. As used herein, natural gelatin means gelatin made from naturally derived collagen.
A chemically synthesized peptide or chemically synthesized gelatin means an artificially synthesized peptide or gelatin. The peptide such as gelatin may be synthesized by solid phase synthesis or liquid phase synthesis, but is preferably solid phase synthesis. Solid-phase synthesis of peptides is known to those skilled in the art. For example, Fmoc group synthesis method using Fmoc group (Fluorenyl-Methoxy-Carbonyl group) as amino group protection, and Boc group (tert-Butyl group) as amino group protection Boc group synthesis method using Oxy Carbonyl group). In addition, the preferable aspect of chemically synthesized gelatin can apply the content described in (1-3) Recombinant gelatin mentioned later in this specification.
Recombinant gelatin will be described later in this specification.

The hydrophilicity value “1 / IOB” value of the biocompatible polymer used in the present invention is preferably from 0 to 1.0. More preferably, it is 0 to 0.6, and still more preferably 0 to 0.4. IOB is an index of hydrophilicity / hydrophobicity based on an organic conceptual diagram representing the polarity / non-polarity of an organic compound proposed by Satoshi Fujita. Details thereof can be found in, for example, “Pharmaceutical Bulletin”, vol.2, 2, pp .163-173 (1954), “Area of Chemistry” vol.11, 10, pp.719-725 (1957), “Fragrance Journal”, vol.50, pp.79-82 (1981), etc. Yes. To put it simply, methane (CH ₄ ) is the source of all organic compounds, and all the other compounds are all methane derivatives, with certain numbers set for their carbon number, substituents, transformations, rings, etc. Then, the score is added to obtain an organic value (OV) and an inorganic value (IV), and these values are plotted on a diagram with the organic value on the X axis and the inorganic value on the Y axis. It is going. The IOB in the organic conceptual diagram means the ratio of the inorganic value (IV) to the organic value (OV) in the organic conceptual diagram, that is, “inorganic value (IV) / organic value (OV)”. For details of the organic conceptual diagram, please refer to “New Organic Conceptual Diagram: Basics and Applications” (Yoshio Koda et al., Sankyo Publishing, 2008). In the present specification, hydrophilicity / hydrophobicity is represented by a “1 / IOB” value obtained by taking the reciprocal of IOB. The smaller the “1 / IOB” value (closer to 0), the more hydrophilic it is.

  By setting the “1 / IOB” value of the polymer used in the present invention within the above range, the hydrophilicity is high and the water absorption is high. It is presumed that the cell structure (mosaic cell mass) of the invention contributes to stabilization of cells and ease of survival.

  When the biocompatible polymer used in the present invention is a polypeptide, the hydrophilicity / hydrophobicity index represented by the Grand average of hydropathicity (GRAVY) value is preferably 0.3 or less and minus 9.0 or more, More preferably, it is 0.0 or less and minus 7.0 or more. Grand average of hydropathicity (GRAVY) values are based on Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins MR, Appel RD, Bairoch A .; Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005) .pp. 571-607 and Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel RD, Bairoch A .; ExPASy: the proteomics server for in-depth protein knowledge and analysis .; Nucleic Acids Res. 31: 3784-3788 (2003).

  By setting the GRAVY value of the polymer used in the present invention within the above range, it has high hydrophilicity and high water absorption, so that it effectively acts to retain nutrient components, and as a result, the cell structure of the present invention. It is presumed to contribute to the stabilization and survival of cells in the body (mosaic cell mass).

(1-2) Crosslinking The biocompatible polymer used in the present invention may be cross-linked or non-cross-linked, but is preferably cross-linked. By using a cross-linked biocompatible polymer, it is possible to obtain an effect of preventing instantaneous degradation when cultured in a culture medium or transplanted to a living body. Common crosslinking methods include thermal crosslinking, crosslinking with aldehydes (eg, formaldehyde, glutaraldehyde, etc.), crosslinking with condensing agents (carbodiimide, cyanamide, etc.), enzyme crosslinking, photocrosslinking, UV crosslinking, hydrophobic interaction, Hydrogen bonds, ionic interactions, etc. are known. In the present invention, it is preferable to use a crosslinking method that does not use glutaraldehyde. In this invention, it is more preferable to use the crosslinking method which does not use aldehydes or a condensing agent. That is, the biocompatible polymer block in the present invention is preferably a biocompatible polymer block that does not contain glutaraldehyde, and more preferably a biocompatible polymer block that does not contain an aldehyde or a condensing agent. . The crosslinking method used in the present invention is more preferably thermal crosslinking, ultraviolet crosslinking, or enzyme crosslinking, and particularly preferably thermal crosslinking.

  When performing cross-linking with an enzyme, the enzyme is not particularly limited as long as it has a cross-linking action between polymer materials, but preferably trans-glutaminase and laccase, most preferably trans-glutaminase can be used for cross-linking. . A specific example of the protein that is enzymatically cross-linked with transglutaminase is not particularly limited as long as it is a protein having a lysine residue and a glutamine residue. The transglutaminase may be derived from a mammal or may be derived from a microorganism. Specifically, transglutaminase derived from a mammal that has been marketed as an Ajinomoto Co., Ltd. activa series, for example, Human-derived blood coagulation factors (Factor XIIIa, Haematologic Technologies, Inc.) such as guinea pig liver-derived transglutaminase, goat-derived transglutaminase, and rabbit-derived transglutaminase manufactured by Oriental Yeast Co., Ltd., Upstate USA Inc., and Biodesign International Etc.).

  The reaction temperature when performing crosslinking (for example, thermal crosslinking) is not particularly limited as long as crosslinking is possible, but is preferably -100 ° C to 500 ° C, more preferably 0 ° C to 300 ° C, and still more preferably. It is 50 degreeC-300 degreeC, More preferably, it is 100 degreeC-250 degreeC, More preferably, it is 120 degreeC-200 degreeC.

(1-3) Recombinant gelatin The recombinant gelatin referred to in the present invention means a polypeptide or protein-like substance having an amino acid sequence similar to gelatin produced by a gene recombination technique. The recombinant gelatin that can be used in the present invention preferably has a repeating sequence represented by Gly-X-Y, which is characteristic of collagen (X and Y each independently represents an amino acid). Here, the plurality of Gly-XY may be the same or different. Preferably, two or more cell adhesion signals are contained in one molecule. As the recombinant gelatin used in the present invention, recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used. For example, EP1014176, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, and the like can be used, but are not limited thereto. A preferable example of the recombinant gelatin used in the present invention is the recombinant gelatin of the following embodiment.

  Recombinant gelatin has excellent biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived, so there is no concern about bovine spongiform encephalopathy (BSE) and excellent non-infectivity. Recombinant gelatin is more uniform than natural ceratin and its sequence is determined, so that strength and degradability can be precisely designed with less blur due to cross-linking and the like.

  The molecular weight of the recombinant gelatin is not particularly limited, but is preferably 2000 or more and 100000 or less (2 kDa or more and 100 kDa or less), more preferably 2500 or more and 95000 or less (2.5 kDa or more and 95 kDa or less), and further preferably 5000 or more and 90000 or less. Or less (5 kDa or more and 90 kDa or less), and most preferably 10000 or more and 90000 or less (10 kDa or more and 90 kDa or less).

  Recombinant gelatin preferably has a repeating sequence represented by Gly-XY characteristic of collagen. Here, the plurality of Gly-XY may be the same or different. In Gly-XY, Gly represents glycine, and X and Y represent any amino acid (preferably any amino acid other than glycine). The sequence shown by Gly-XY, which is characteristic of collagen, is a very specific partial structure in the amino acid composition and sequence of gelatin / collagen compared to other proteins. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, it is one in three repeats. Glycine is the simplest amino acid, has few constraints on the arrangement of molecular chains, and greatly contributes to the regeneration of the helix structure upon gelation. The amino acids represented by X and Y are rich in imino acids (proline, oxyproline) and preferably account for 10% to 45% of the total. Preferably, 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acid sequence of the recombinant gelatin is a Gly-XY repeating structure.

  In general gelatin, polar amino acids have a charged amino acid and an uncharged one in a ratio of 1: 1. Here, the polar amino acid specifically refers to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine and arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. Refers to threonine and tyrosine. In the recombinant gelatin used in the present invention, the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituting amino acids. In addition, the proportion of uncharged amino acids in the polar amino acid is preferably 5% or more and less than 20%, preferably less than 10%. Furthermore, it is preferable that any one amino acid, preferably two or more amino acids among serine, threonine, asparagine, tyrosine and cysteine are not included in the sequence.

  In general, the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology” published by Nagai Publishing Co., Ltd., Vol. 9, No. 7 (1990), page 527). The recombinant gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule. Specific sequences include RGD sequences, LDV sequences, REDV sequences, YIGSR sequences, PDSGR sequences, RYVVLPR sequences, LGITIPG sequences, RNIAEIIKDI sequences, which are expressed in one-letter amino acid notation in that many types of cells adhere. , IKVAV sequences, LRE sequences, DGEA sequences, and HAV sequences are preferred. More preferred are RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence and HAV sequence, and particularly preferred is RGD sequence. Of the RGD sequences, an ERGD sequence is preferred. By using recombinant gelatin having a cell adhesion signal, the amount of cell substrate produced can be improved. For example, in the case of cartilage differentiation using mesenchymal stem cells as cells, production of glycosaminoglycan (GAG) can be improved.

Regarding the arrangement of RGD sequences in the recombinant gelatin used in the present invention, it is preferable that the number of amino acids between RGDs is not uniform between 0 and 100, preferably between 25 and 60.
The content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 in one protein molecule from the viewpoint of cell adhesion / proliferation. Most preferably, it is 12.

  In the recombinant gelatin used in the present invention, the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%. When the recombinant gelatin contains 350 or more amino acids, it is preferred that each stretch of 350 amino acids contains at least one RGD motif. The ratio of RGD motif to the total number of amino acids is more preferably at least 0.6%, more preferably at least 0.8%, more preferably at least 1.0%, more preferably at least 1.2%. And most preferably at least 1.5%. The number of RGD motifs in the recombinant peptide is preferably at least 4, more preferably 6, more preferably 8, more preferably 12 or more and 16 or less per 250 amino acids. A ratio of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, a gelatin of 251 amino acids must contain at least two RGD sequences to meet the 0.4% feature. Preferably, the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains the RGD sequence. As a further aspect of the recombinant gelatin of the present invention, it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less.

  Recombinant gelatin may be partially hydrolyzed.

Preferably, the recombinant gelatin used in the present invention is represented by the formula: A-[(Gly-XY) _n ] _m -B. Each of n Xs independently represents any of amino acids, and each of n Ys independently represents any of amino acids. m is preferably 2 to 10, and preferably 3 to 5. n is preferably 3 to 100, more preferably 15 to 70, and most preferably 50 to 65. A represents any amino acid or amino acid sequence, and B represents any amino acid or amino acid sequence.

More preferably, the recombinant gelatin used in the present invention has the formula: Gly-Ala-Pro-[(Gly-XY) ₆₃ ] ₃ -Gly (wherein 63 Xs independently represent any of amino acids). And 63 Y's each independently represent any of the amino acids, wherein 63 Gly-XY may be the same or different.

  It is preferable to bind a plurality of naturally occurring collagen sequence units to the repeating unit. The naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, or type V collagen. More preferred is type I, type II, or type III collagen. According to another form, the collagen origin is preferably human, bovine, porcine, mouse or rat, more preferably human.

  The isoelectric point of the recombinant gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.

Preferably, the recombinant gelatin is not deaminated.
Preferably, the recombinant gelatin has no telopeptide.
Preferably, the recombinant gelatin is a substantially pure polypeptide prepared with a nucleic acid encoding an amino acid sequence.

Particularly preferably as the recombinant gelatin used in the present invention,
(1) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(2) A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility; or (3) described in SEQ ID NO: 1 A peptide having an amino acid sequence and an amino acid sequence having a sequence identity of 80% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) and having biocompatibility;
It is.

  The “one or several” in the “amino acid sequence in which one or several amino acids have been deleted, substituted or added” is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. Means, particularly preferably 1 to 3.

  The recombinant gelatin used in the present invention can be produced by a genetic recombination technique known to those skilled in the art. For example, in EP1014176A2, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, etc. It can be produced according to the method described. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is introduced into an appropriate host to produce a transformant. . Recombinant gelatin is produced by culturing the obtained transformant in an appropriate medium. Therefore, the recombinant gelatin used in the present invention can be prepared by recovering the recombinant gelatin produced from the culture. .

(1-4) Biocompatible polymer block In the present invention, a block (lumb) made of the above-described biocompatible polymer is used.
The shape of the biocompatible polymer block in the present invention is not particularly limited. For example, amorphous, spherical, particulate (granule), powder, porous, fiber, spindle, flat and sheet, preferably amorphous, spherical, particulate (granule), powder And porous. An indeterminate shape indicates that the surface shape is not uniform, for example, an object having irregularities such as rocks. In addition, illustration of said shape is not different, respectively, For example, it may become an indefinite form as an example of the subordinate concept of a particulate form (granule).

  The size of one biocompatible polymer block in the present invention is not particularly limited, but is preferably 1 μm or more and 1000 μm or less, more preferably 10 μm or more and 1000 μm or less, more preferably 10 μm or more and 700 μm or less, Preferably they are 10 micrometers or more and 300 micrometers or less, More preferably, they are 10 micrometers or more and 200 micrometers or less, More preferably, they are 20 micrometers or more and 200 micrometers or less, More preferably, they are 20 micrometers or more and 150 micrometers or less, More preferably, they are 50 micrometers or more and 110 micrometers or less. Setting the size of one biocompatible polymer block within the above range is preferable from the viewpoint that the tubular structure has higher molecular permeability. The size of one biocompatible polymer block does not mean that the average value of the sizes of a plurality of biocompatible polymer blocks is in the above range. It means the size of each biocompatible polymer block obtained by sieving the block.

  The size of one block can be defined by the size of the sieve used to separate the blocks. For example, the block remaining on the sieve when the block passed through the sieve of 180 μm and passed through the sieve of 106 μm can be a block having a size of 106 to 180 μm. Next, a block having a size of 53 to 106 μm can be formed by passing through a 106 μm sieve and leaving the passed block on the 53 μm sieve. Next, the block remaining on the sieve when passing through the sieve of 53 μm and passing the passed block through the sieve of 25 μm can be a block having a size of 25 to 53 μm.

(1-5) Bioaffinity polymer block production method The bioaffinity polymer block production method is not particularly limited. For example, a bioaffinity polymer porous material is removed from a pulverizer (such as a new power mill). ) Can be used to obtain an amorphous biocompatible polymer block which is an example of a granular form.

  When producing a porous body of a biocompatible polymer, freezing in which the liquid temperature at the highest liquid temperature in the solution (internal maximum liquid temperature) is not more than “solvent melting point −3 ° C.” in an unfrozen state By including the process, the ice formed will be spherical. Through this step, the ice is dried to obtain a porous body having spherical isotropic pores (spherical holes). It is formed by freezing without including a freezing step in which the liquid temperature at the highest liquid temperature in the solution (internal maximum liquid temperature) becomes “solvent melting point −3 ° C.” or higher in an unfrozen state. Ice becomes pillar / flat. When the ice is dried through this step, a porous body having columnar or plate-like pores (column / plate hole) that is long on one or two axes is obtained.

In the present invention, preferably,
In the biocompatible polymer solution, the internal maximum liquid temperature which is the liquid temperature of the highest liquid temperature in the solution is 3 ° C. lower than the solvent melting point in an unfrozen state (“solvent melting point −3 ° C.”). Step a for freezing by freezing treatment; and Step b for lyophilizing the frozen biocompatible polymer obtained in Step a above:
A biocompatible polymer block can be produced by a method comprising:
More preferably, in the present invention, a biocompatible polymer block in the form of granules can be produced by pulverizing the porous body obtained in the step b.

  More preferably, in step a, the biocompatible polymer solution is a temperature at which the internal maximum liquid temperature, which is the liquid temperature at the highest temperature in the solution, is 7 ° C. lower than the solvent melting point in an unfrozen state. (“Solvent melting point−7 ° C.”) or below can be frozen by a freezing treatment.

(2) Cells The cells used in the present invention are fibroblasts, vascular endothelial cells and smooth muscle cells, more preferably dermal fibroblasts, umbilical vein vascular endothelial cells and bladder smooth muscle cells. In the present invention, as long as fibroblasts, vascular endothelial cells and smooth muscle cells are used, cells other than the above three types may be used, or only the above three types of cells may be used. The cell to be used is preferably a vertebrate cell, and particularly preferably a human cell. The origin of the cell may be either an autologous cell or an allogeneic cell.
The fibroblasts, vascular endothelial cells and smooth muscle cells may be normal human-derived (adult or child) primary cells or established cells.

  Fibroblasts such as skin fibroblasts can be detected by, for example, CD90 positive, which is a surface antigen specific for fibroblasts. CD is an abbreviation for Cluster Designation.

Vascular endothelial cells such as umbilical vein vascular endothelial cells can be detected by, for example, von Willebrand factor (vWF) positive, CD31 positive, Dil-Ac-LDL uptake positive, or smooth muscle α-actin negative. Dil-Ac-LDL represents Dil-labeled acetyl low density lipoprotein. Dil represents the fluorescent probe 1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethol-indocarbocyanine perchlorate.
Vascular endothelial cells such as bladder smooth muscle cells can be detected by, for example, smooth muscle α-actin positive or CD90 negative.

(3) Cell structure In the present invention, a plurality of biocompatible polymer blocks are mosaic-shaped in the gaps between a plurality of cells using the biocompatible polymer block and the predetermined cells. A cell structure is produced by dimensional arrangement. By arranging the biocompatible polymer block and the cells in a three-dimensional mosaic manner, a cell structure in which the cells are uniformly present in the structure is formed, and the medium from the outside to the inside of the cell structure is formed. Delivery of nutrients such as ingredients becomes possible.

  In the cell structure used in the present invention, a plurality of biocompatible polymer blocks are arranged in the gaps between the plurality of cells. Therefore, it is not necessary to be a closed space, as long as it is sandwiched between cells. Note that there is no need for a gap between all cells, and there may be a place where the cells are in contact with each other. The gap distance between the cells via the biocompatible polymer block, that is, the gap distance when selecting a cell and a cell that is present at the shortest distance from the cell is not particularly limited. The size is preferably the size of the polymer block, and the preferred distance is also within the preferred size range of the biocompatible polymer block.

  In addition, the biocompatible polymer block is sandwiched between cells, but there is no need for cells between all the biocompatible polymer blocks, and the biocompatible polymer blocks are in contact with each other. There may be. The distance between the biocompatible polymer blocks via the cells, that is, the distance when the biocompatible polymer block and the biocompatible polymer block existing at the shortest distance from the biocompatible polymer block are selected are particularly Although not limited, it is preferably the size of a cell mass when one to several cells are used, for example, 10 μm or more and 1000 μm or less, preferably 10 μm or more and 100 μm or less. More preferably, it is 10 μm or more and 50 μm or less.

  In addition, in this specification, the expression “is uniformly present” such as “a cell structure in which cells are uniformly present in the structure” is used, but does not mean complete uniformity, This means that nutrients such as medium components can be delivered from the outside to the inside of the cell structure.

  The thickness or diameter of the cell structure can be set to a desired thickness, but the lower limit is preferably 215 μm or more, more preferably 400 μm or more, and most preferably 500 μm or more. The upper limit of the thickness or diameter is not particularly limited, but the general range for use is preferably 3 cm or less, more preferably 2 cm or less, and even more preferably 1 cm or less. The range of the thickness or diameter of the cell structure is preferably 400 μm or more and 3 cm or less, more preferably 500 μm or more and 2 cm or less, and further preferably 500 μm or more and 1 cm or less. By making the thickness or diameter of the cell structure within the above range, it becomes easy to produce a tubular structure using the cell structure.

  In the cell structure, preferably, a region composed of a biocompatible polymer block and a region composed of cells are arranged in a mosaic pattern. In addition, the “thickness or diameter of the cell structure” in this specification indicates the following. When a certain point A in the cell structure is selected, the length of the line segment that divides the cell structure so that the distance from the outside of the cell structure becomes the shortest in a straight line passing through the point A is shown. Minute A. A point A having the longest line segment A is selected in the cell structure, and the length of the line segment A at that time is defined as the “thickness or diameter of the cell structure”.

  The ratio of the cells and the biocompatible polymer block in the cell structure is not particularly limited, but preferably the ratio of the biocompatible polymer block per cell is preferably 0.0000001 μg or more and 1 μg or less. Is 0.000001 μg to 0.1 μg, more preferably 0.00001 μg to 0.01 μg, and most preferably 0.00002 μg to 0.006 μg. By setting the ratio of the cell and the biocompatible polymer block within the above range, the cells can be made to exist more uniformly, and the ratio of the volume of the biocompatible polymer block to the volume of the cell structure and the cell structure The ratio of the cell volume to the volume of the cell can be within the range defined in the present invention. By setting the lower limit to the above range, the effect of the cells can be exerted when used for the above-mentioned applications, and by setting the upper limit to the above range, the components in the biocompatible polymer block that are optionally present are cells. Can supply. Here, the component in the biocompatible polymer block is not particularly limited, and examples thereof include components contained in the medium described later.

  The cell structure may contain an angiogenic factor. Here, preferred examples of the angiogenic factor include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and the like. Although the manufacturing method of the cell structure containing an angiogenic factor is not particularly limited, for example, it can be manufactured by using a biocompatible polymer block impregnated with an angiogenic factor. From the viewpoint of promoting angiogenesis, the cell structure of the present invention preferably contains an angiogenic factor.

(4) Method for producing cell structure A cell structure can be produced by mixing a biocompatible polymer block and cells (fibroblasts, vascular endothelial cells and smooth muscle cells). More specifically, the cell structure can be produced by alternately arranging biocompatible polymer blocks and cells. The production method is not particularly limited, but is preferably a method of seeding cells after forming a biocompatible polymer block. Specifically, a cell structure can be produced by incubating a mixture of a biocompatible polymer block and a cell-containing culture solution. For example, cells and a biocompatible polymer block prepared in advance are arranged in a mosaic in a container and in a liquid held in the container. As a means of arrangement, it is preferable to promote or control the formation of a mosaic array composed of cells and a biocompatible substrate by using natural aggregation, natural dropping, centrifugation, and stirring.

  The container used is preferably a container made of a low cell adhesion material or a cell non-adhesive material, more preferably a container made of polystyrene, polypropylene, polyethylene, glass, polycarbonate, or polyethylene terephthalate. The shape of the bottom surface of the container is preferably a flat bottom type, a U shape, or a V shape.

(5) Tubular structure The manufacturing method of the tubular structure of the present invention will be described later.
A schematic diagram of an example of the tubular structure of the present invention is shown in FIG. In FIG. 1, an inner diameter 21, an outer diameter 22, and a length 23 of the tubular structure are shown. The tubular structure of the present invention has a structure having a substantially columnar cavity inside a substantially columnar structure. However, the shape of the cross section is not limited to a strict circle, and may be a shape similar to a circle such as an ellipse.

The magnitude | size of the tubular structure of this invention is not specifically limited, The tubular structure of a desired magnitude | size can be designed according to a use.
The inner diameter of the tubular structure of the present invention is preferably 1 mm or more and less than 6 mm, more preferably 1 mm or more and 5 mm or less, and further preferably 1 mm or more and 3 mm or less in consideration of the use as an artificial blood vessel.
The outer diameter of the tubular structure of the present invention is preferably 3 mm or more and 10 mm or less, more preferably 3 mm or more and 8 mm or less, and further preferably 3 mm or more and 5 mm or less in consideration of the use as an artificial blood vessel.
The difference between the inner diameter and the outer diameter is preferably 1 mm or more and 9 mm or less, and more preferably 2 mm or more and 5 mm or less from the viewpoint of the strength of the tubular structure.
The length of the tubular structure of the present invention is preferably 3 mm or more and 300 mm or less, and more preferably 3 mm or more and 150 mm or less in consideration of the use as an artificial blood vessel.
Here, the inner diameter and the outer diameter mean the inner diameter and the outer diameter when the cross section of the tubular structure of the present invention is approximated to a circle.

(6) Use of tubular structure The tubular structure of the present invention can be used as an artificial blood vessel, an artificial urinary tract, or an artificial digestive tract, and can be preferably used as an artificial blood vessel. Specifically, the cell structure of the present invention can be used for the purpose of transplantation at a site where an artificial blood vessel, an artificial ureter, or an artificial digestive tract needs to be transplanted, for example.
As an implantation method, an incision, an endoscope, or the like can be used.

  According to the present invention, there is provided a transplantation method including the step of transplanting a tubular structure to a patient in need of transplantation of an artificial blood vessel. In the transplantation method of the present invention, the above-described tubular structure of the present invention is used. Suitable ranges of the cell structure and the tubular structure are the same as described above.

  The present invention further provides the use of the tubular structure of the present invention for the manufacture of artificial blood vessels. Suitable ranges of the cell structure and the tubular structure are the same as described above.

[Method for producing cell structure]
The present invention further provides a cell structure comprising a biocompatible polymer block and cells, wherein a plurality of the polymer blocks are disposed in a gap between the plurality of cells, and having an arbitrary shape. Regarding the method. The method for producing a cell structure according to the present invention includes a biocompatible polymer block and a cell in the thread or needle of the support including a substrate and a thread or needle. A plurality of the polymer blocks are arranged in the gaps between the cells, the plurality of cell structures are penetrated, and the cell structure is formed by the plurality of cell structures. And the step A of culturing the cell structure arranged in the step A and fusing adjacent cell structures together. The method for producing a cell structure according to the present invention may further include a step C of drawing out the cell structure formed in an arbitrary shape in the step B from the filamentous body or the needle-shaped body.

  The method for producing a cell structure according to the present invention uses a cell structure including a biocompatible polymer block and cells, wherein a plurality of the polymer blocks are disposed in a space between the plurality of cells. Except for this, it can be carried out according to the method described in Japanese Patent No. 4517125.

  A schematic view of an example of the support used in the present invention is shown in FIG. In FIG. 2, the support 10 includes a substrate 11 having an arbitrary shape, and a filament or needle 12 on the substrate 11. FIG. 2 shows an example of a mode in which the filaments or needles are arranged substantially in the normal direction of the base surface of the substrate. The substrate 11 and the thread-like body or needle-like body 12 as a whole may be composed of separate parts and fixed, or may be integrally made of, for example, a thermoplastic resin. The number of filaments or needles that the support has is one or more, and any desired number of filaments or needles can be used. In this specification, “substantially normal direction” means an arbitrary angle in which the longitudinal direction of the filamentous body or needle-like body is about 90 degrees (for example, 80 degrees to 100 degrees) with respect to the base surface of the substrate 11. , And preferably 90 degrees. In another embodiment, the substrate 11 may further include a sheet that covers the surface of the substrate on which the filamentous body or needle-like body is present (see FIG. 1B of Japanese Patent No. 4517125). The surface area of the sheet may be smaller, the same or larger than the surface area of the base surface of the substrate 11, and preferably covers the base surface of the region where the filamentous body or the needle-like body exists.

  As another aspect, the filaments or needles may be arranged in a non-normal direction (for example, a direction having an angle from the normal direction) instead of the normal direction. The angle of the “non-normal direction” thread or needle can be appropriately selected within a range of 1 to 79 degrees with respect to the support 10, for example, 10 degrees, 20 degrees, 30 degrees, and 40 degrees. , 50 degrees, 60 degrees, 70 degrees, and the like. In addition, the “non-normal direction” thread or needle may extend linearly from the support in a certain direction, or may extend non-linearly, such as part of a circular or elliptical arc. (FIG. 1C of Japanese Patent No. 4517125). The shape of the thread-like body or needle-like body existing on one support does not need to be unified with one mold, and various types of thread-like bodies or needle-shaped bodies can be used in combination (Patent No. 1). FIG. 1C) of No. 4517125. These modes include a support body that is a combination of a filamentous body or needle in the normal direction and a thread or needle having a certain angle in the non-normal direction, a thread or needle in the normal direction, and an arc. A support combining a thread or needle having a locus corresponding to a part thereof, and a thread or needle in a normal direction and a thread or needle having a certain angle in a non-normal direction and an arc. Examples include, but are not limited to, a support combined with a thread or needle having a locus corresponding to a part. For example, the filamentous body or needle-like body can be curved, and arranged on the circumference to form an arch, and the cell structure can be penetrated and fused along the arch. Further, if the filamentous body or needle-like body is linear, it can be formed into a conical shape, a hollow shape, or a pyramid shape depending on the arrangement. By using a combination of a plurality of types of filamentous bodies or needlelike bodies, it is possible to form a cell structure having a complicated shape.

  The needle-like body or the needle-like body and the sheet are all preferably made of a non-cell-adhesive material. The substrate is also preferably non-cell-adhesive, but when a sheet is used as the support, the cell structure is not in direct contact with the substrate, so the material is not particularly limited. Cell non-adhesive means the property of preventing cells from adhering to the wall surface via an extracellular adhesion factor, such as a material coated with a substance that provides a cell non-adhesive function (for example, fluorine) Has the above properties. In a preferred embodiment, the thread or needle is made of polypropylene, nylon, or stainless steel. In another preferred embodiment, the sheet is a fluorine-processed or polyhydroxyethylmethacrylate polymer (polyhydroxyethyl methacrylate polymer) process (poly-HEMA process).

  Thread or needle-like body and sheet are made of Teflon (registered trademark), poly-HEMA, acrylic plate, vinyl chloride plate, polyester resin plate, polycarbonate plate, etc., PP (polypropylene), ABS (acrylonitrile butadiene styrene) Engineering plastics such as PE (polyethylene), POM (polyacetal), PC (polycarbonate), PEEK (polyether ether ketone), MCN (monomer casting nylon), 6N (6 nylon), 66N (66 nylon) may be used. In addition to these materials, materials with reduced cell adhesion may be used, but are not limited thereto. The support may be made of a bioabsorbable material. However, if a bioabsorbable material is used, there is a possibility that a decomposition product or an insoluble residue may remain and cause toxicity. The above materials are preferred instead of the absorbent material.

  The thread-like body or needle-like body is a rod-like body for penetrating the cell structure in a so-called skewered shape, and each thread-like body or needle-like body has a thread-like body or needle-like shape adjacent to the penetrated cell structure. They are positioned relative to each other so that they can be brought into contact with and fused with cellular structures that have penetrated the body. The filaments or needles may be arranged regularly, for example, in a lattice pattern, or randomly. The interval between the filamentous bodies or needle-like bodies may vary depending on the size of the cell structure passing therethrough, but is preferably defined by a length of approximately 100% to 110% of the diameter of the cell structure. For example, when the diameter of the cell structure is 1 mm, the distance between the filamentous bodies or needle-like bodies is preferably about 1 mm to 1.1 mm.

  The diameter of the cross-section of the filament or needle can take any value as long as it does not destroy the cell structure and prevents the cell structure from fusing. Moreover, in one embodiment, a needle-like body is a cone which contact | connects the bottom face of a cone to the base face of a board | substrate (FIG. 1D of patent 4517125). The sheet has a hole or a mesh shape so that the filament or needle-like body arranged substantially in the normal direction of the base surface of the substrate can pass through the sheet. The substrate and the filament or needle It can be removed from the body. A specific embodiment in which the acicular body is conical (FIG. 1D of Japanese Patent No. 4517125) is preferred in that it can facilitate the removal of the sheet from the acicular body. The support is mainly used for temporary fixation until the cell structures are fused to obtain a cell structure having a desired shape. In some embodiments, the cell structure can be maintained on the support and used as an organ simulator or the like. In this case, the support may or may not be pulled out from the cell structure.

  In another embodiment, the filamentous body may be, for example, a suture thread. Preferably, a suture thread with a needle can be used as the filamentous body. For example, by fixing one end of the thread on the substrate and making the other end have a sharp shape (for example, a needle), it is easier to penetrate the cell structure through the thread (Patent No. 1). No. 4517125, FIG. 1E). It is also possible to arrange the cell structure by passing the cell structure through a thread and fixing the thread in a tensioned state (FIG. 1F of Japanese Patent No. 4517125). A plurality of predetermined cell structures are sequentially penetrated into a thread having one end fixed on the first substrate to form a skewered state, and then the tip of the thread used to penetrate the cell structure is second It can also be fixed on the substrate. Also, a thread that is sufficiently long relative to the distance between the first substrate and the second substrate is used, and the thread that has penetrated the cell structure is several times between the first substrate and the second substrate. It is also possible to reciprocate and fix the gap so that it can come into contact with adjacent cell structures.

  According to the present invention, a plurality of cell structures comprising a biocompatible polymer block and cells, wherein a plurality of the polymer blocks are disposed in gaps between the plurality of cells, It arrange | positions in the arbitrary positional spaces on the support body. When arranging a plurality of cell structures in an arbitrary position space on the support, the plurality of cell structures are made to penetrate through the filaments or needles on the support. This step can be performed, for example, by directing the tip of the pipette containing the cell structure toward the tip of the needle-like body and pushing out the cell structure by applying pressure from the side opposite to the tip of the pipette (Patent No. 1). FIG. 2A) of No. 4517125. The extruded cell structure is pierced into the needle-like body and fixed at a predetermined position. Alternatively, in another embodiment, this step can be performed by penetrating the cell structure from above the needle-like body using a small robot arm carrying the cell structure (Japanese Patent No. 4517125). FIG. 2B). It is also possible to fix the cell structure with tweezers or the like and allow the thread (preferably a thread with a needle) to penetrate the cell structure. However, the method of the present invention is not limited to such steps. The cell structure can be penetrated so that a plurality of cell structures are in contact with one filament or needle (FIG. 2C of Japanese Patent No. 4517125). By contacting the cell structures, the cell structures can be fused in the vertical direction (vertical direction (for example, z direction)). In addition, each filamentous body or needle-like body is positioned at such an interval that it can come into contact with an adjacent cellular structure when penetrating the cellular structure, so that the lateral direction (horizontal direction and depth direction (for example, , X direction and y direction)). Therefore, a cell structure having an arbitrary shape can be constructed on the support through the fusion of these cell structures.

  A cell structure having an arbitrary shape can be obtained by collecting the cell structure fused in contact with the above. Recovery of the cell structure can be achieved by a process of pulling out the fused cell structure from the filament or needle. Pulling out the cell structure may be accomplished by pulling out the filament or needle from the fused cell structure directly fixed with tweezers, or by removing the sheet from the support. Sometimes achieved. The removal of the sheet may be performed by pulling out the support from the fixed sheet, or may be performed by separating the sheet from the fixed support. A method comprising these series of steps provides a cell structure having any shape spatially arranged as desired.

[Method for producing tubular structure]
The cell structure (mosaic cell mass) obtained by the above (4) cell structure production method is, for example,
(A) fusing cell structures (mosaic cell masses) together, or (b) increasing the volume under differentiation medium or growth medium,
The tubular structure of the present invention can be manufactured by such a method. The fusion method and the volume increase method are not particularly limited. In the case of fusing cell structures, for example, a plurality of biocompatible polymer blocks and a plurality of cells are included, and a part or all of the plurality of gaps formed by the plurality of cells are combined. Alternatively, a plurality of cell structures in which a plurality of the biocompatible polymer blocks are arranged can be fused.

  “Biocompatible polymer block (type, size, etc.)”, “cell”, “gap between cells”, “obtained cell structure (size, etc.)” according to the method for producing a tubular structure of the present invention A suitable range such as “ratio of cells and biocompatible polymer block” is the same as described above in the present specification.

  The thickness or diameter of each cell structure before fusion is preferably 10 μm or more and 1 cm or less, more preferably 10 μm or more and 2000 μm or less, still more preferably 15 μm or more and 1500 μm or less, and most preferably 20 μm or more and 1300 μm or less. The thickness or diameter after fusion is preferably 400 μm or more and 3 cm or less, more preferably 500 μm or more and 2 cm or less, and further preferably 720 μm or more and 1 cm or less.

The fusion of a plurality of cell structures is preferably performed by the method described in [Method for producing cell structures] in the present specification.
That is, the fusion includes a biocompatible polymer block and cells in the thread or needle of the support including a substrate and a thread or needle, and a gap between the cells. Step A1 of arranging a cell structure so that a plurality of cell structures in which a plurality of the polymer blocks are arranged are penetrated and a tubular structure is formed by the plurality of cell structures. It is preferable to carry out by the method including the step B1 of culturing the cell structures arranged in the step A1 and fusing adjacent cell structures. The cells used here include fibroblasts, vascular endothelial cells and smooth muscle cells.

  The step A1 of arranging the cell structure so that the shape of the tubular structure is formed by the plurality of cell structures is performed by using the support including the substrate and the filamentous body or the needle-shaped body, A tubular structure of a cell structure can be formed by disposing it at a substantially circumferential position. As a first example of the method of arranging the cell structures approximately in a circumferential shape, for example, a support having needle-like bodies (9 vertical × 9 horizontal) in the arrangement shown in FIG. 4 is used. One example of the needle-like body on the support is a penetration of the cell structure with respect to a part of the needle-like body (that is, a needle-like body substantially corresponding to a position on the circumference). In FIG. 4, in the first and ninth stages from the top, the cell structures are passed through the five needles 32 from the left to the third to the seventh, respectively, and in the second and eighth stages from the top. Pass through the cell structure through 7 needles 32 from the 2nd to the 8th from the left, respectively, and in the 3rd and 7th tiers from the top to the 3rd from the left and 7th to 9th from the left, respectively. The six needle-like bodies 32 are passed through the cell structure, and the fourth to sixth stages from the top are the first, second, seventh and ninth four needle-like bodies 32 from the left. The cell structure is penetrated. On the other hand, the needle-like body 31 is a needle-like body that does not penetrate the cell structure.

  As a second example of the method of arranging the cell structures approximately in a circumferential shape, a filamentous body or a needle-like body is disposed on a substantially circumferential surface on the substrate, and these filamentous body or needle-like body is disposed. For example, the cell structure can be penetrated through the body.

  By culturing a plurality of cell structures penetrating the filamentous body or needle-like body, adjacent cell structures can be fused together, whereby a tubular structure of the cell structure is formed. .

  Furthermore, the present invention includes a step C1 of pulling out the cell structure formed in the shape of the tubular structure in step B1 from the filamentous body or needle-like body, and a step D1 of culturing the cell structure obtained in step C1. be able to. By culturing the cell structure for a predetermined period, the strength of the tubular structure can be increased.

The cell structure can be cultured by a conventional method known to those skilled in the art. For example, in a medium prepared by mixing a medium dedicated to normal human skin fibroblasts (NHDF), a medium dedicated to human umbilical vein endothelial cells (HUVEC), and a medium dedicated to human bladder smooth muscle cells (HBdSMC), Can be cultured under conditions of 5% CO ₂ .

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

[Example 1] Recombinant peptide (recombinant gelatin)
The following CBE3 was prepared as a recombinant peptide (recombinant gelatin) (described in International Publication No. WO2008 / 103041).
CBE3:
Molecular weight: 51.6 kD
Structure: GAP [(GXY) ₆₃ ] ₃ G
Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33%
Almost 100% of amino acids are GXY repeating structures. The amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine. CBE3 has an ERGD sequence.
Isoelectric point: 9.34
GRAVY value: -0.682
1 / IOB value: 0.323
Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 of International Publication WO2008 / 103041; however, the suffix X is corrected to “P”)
GAP (GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGAPG

[Example 2] Production of recombinant peptide porous body
[PTFE thickness / cylindrical container]
A cylindrical cup-shaped container made of polytetrafluoroethylene (PTFE) having a bottom surface thickness of 3 mm, a diameter of 51 mm, a side surface thickness of 8 mm, and a height of 25 mm was prepared. When the cylindrical cup-shaped container made of PTFE has a curved surface as a side surface, the side surface is closed with 8 mm PTFE, and the bottom surface (flat plate shape) is also closed with 3 mm PTFE. On the other hand, the upper surface has an open shape. Therefore, the inner diameter of the cylindrical cup-shaped container is 43 mm. Hereinafter, this container is referred to as a PTFE thick / cylindrical container.

The CBE3 aqueous solution was poured into a PTFE thick / cylindrical container, and the CBE3 aqueous solution was cooled from the bottom using a cooling shelf in a vacuum freeze dryer (TF5-85ATNNN: manufactured by Takara Seisakusho). The final concentration of the CBE3 aqueous solution is 4% by mass, and the amount of the aqueous solution is 8 mL. The shelf temperature is set to -10 ° C, cooled to -10 ° C for 1 hour, then -20 ° C for 2 hours, further -40 ° C for 3 hours, and finally frozen at -50 ° C for 1 hour. It was. The frozen product thus obtained was then subjected to vacuum drying at −20 ° C. for 24 hours after the shelf temperature was returned to the −20 ° C. setting, and after 24 hours, the shelf temperature was increased to 20 ° C. It was raised and vacuum drying was further carried out at 20 ° C. for 48 hours until the degree of vacuum was sufficiently lowered (1.9 × 10 ⁵ Pa), and then it was taken out from the vacuum freeze dryer. The porous body was obtained by the above.

  When the porous body is produced, each aqueous solution is cooled from the bottom surface, so that the water surface temperature at the center of the circle is hardly cooled. Therefore, since the water surface portion at the center of the circle has the highest liquid temperature in the solution, the temperature of the water surface at the center of the circle was measured. Hereinafter, the liquid temperature of the water surface portion at the center of the circle is referred to as the internal maximum liquid temperature.

[Example 3] Measurement of internal maximum liquid temperature in freezing step FIG. 3 shows a temperature profile when a solvent is frozen. After passing through an unfrozen state below the melting point, solidification heat is generated and the temperature starts to rise, and ice formation actually begins at this stage. Thereafter, the temperature passed around 0 ° C. for a certain time, and at this stage, a mixture of water and ice was present. At the end, the temperature starts to drop again from 0 ° C. At this stage, the liquid portion disappears and ice is formed. The temperature being measured is the solid temperature inside the ice, not the liquid temperature. As described above, by looking at the internal maximum liquid temperature at the moment when the heat of solidification occurs, it can be determined whether the internal maximum liquid temperature has been frozen after passing through the “solvent melting point −3 ° C.” in an unfrozen state.

  The internal maximum liquid temperature in the unfrozen state at the moment when the heat of solidification was generated was −8.8 ° C. When the internal maximum liquid temperature at the moment when the heat of solidification is generated is seen, it can be seen that the internal maximum liquid temperature is not more than “solvent melting point−3 ° C.” in an unfrozen state.

[Example 4] Preparation of recombinant peptide block (pulverization and crosslinking of porous material)
The CBE3 porous material obtained in Example 2 was pulverized with New Power Mill (manufactured by Osaka Chemical Co., Ltd., New Power Mill PM-2005). The pulverization was performed by pulverization for 5 minutes in total for 1 minute × 5 times at the maximum rotation speed. The obtained pulverized product was sized with a stainless steel sieve to obtain CBE3 blocks in the form of granules of 25 to 53 μm, 53 to 106 μm, and 106 μm to 180 μm. Thereafter, thermal crosslinking was performed at 160 ° C. under nitrogen (crosslinking time was 8 to 48 hours) to obtain a recombinant peptide block. Hereinafter, all blocks having a size of 53 to 106 μm were used.

[Comparative Example 1] Production of spheroids Production of spheroids containing only cells was performed as follows. Normal human skin fibroblasts (NHDF) (10000 cells / well), human umbilical vein endothelial cells (HUVEC) (8000 cells / well) and human bladder smooth muscle cells (HBdSMC) (2000 cells / well) A suspension containing 200 μl / well of an equal amount of the above-mentioned dedicated medium (below) was prepared and plated in each well of PrimeSurface 96-well plate (Sumitomo Bakelite). The culture was performed at 37 ° C. for 3 days in a CO ₂ incubator.

Each cell and its dedicated medium were obtained from TAKARA BIO INC.
For NHDF, see http://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100007858.
Normal human skin fibroblasts (adult): Normal Human Dermal Fibroblasts (NHDF), adult donor, Takara, C-12302
Special medium is Fibroblast Growth Medium Kit, Takara, C-23110
For HUVEC, see http://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100007840.
Human Umbilical Vein Endothelial Cells (HUVEC), Takara, C-12200
Dedicated medium is Endothelial Cell Growth Medium 2 Kit, Takara, C-22111
For HBdSMC, see http://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100007871.
Human bladder smooth muscle cells (HBdSMC), Takara, C-12571
The special medium is Smooth Muscle Cell Growth Medium 2 Kit, Takara, C-22062

  The spheroid prepared by the above method is laminated on a support having the arrangement shown in FIG. 2 and FIG. 4 using a bio 3D printer “Regenova” (registered trademark) (Cifu Uses Co., Ltd.), and then the same composition as the spheroid production For 3 days. In FIG. 4, the diameter of one needle-like body is 180 μm, and the interval between the needle-like bodies is 400 μm.

  In the tubular structure for tensile strength test, 48 needle-shaped bodies (reference numeral 32 in FIG. 4) of the ring-shaped needle-shaped bodies arranged in 9 × 9 are prepared for the production of one tubular structure. The spheroid was penetrated. Further, eight spheroids (that is, eight layers) were passed through each needle-like body. That is, since eight layers of 48 spheroids are provided for each tubular structure, the total number of spheroids is 384. The manufactured tubular structure had an outer diameter of 3.6 mm, an inner diameter of 2 mm, and a length of 3.2 mm.

  In the tubular structure for the water permeability test, for the production of one tubular structure, the ring-shaped 48 needle-shaped bodies (reference numeral 32 in FIG. 4) among the needle-shaped bodies arranged in 9 × 9. The spheroid was penetrated. Further, 18 spheroids (that is, 18 layers) were passed through each needle-like body. That is, since one layer of 48 spheroids per layer is provided for one tubular structure, the total number of spheroids is 864. The manufactured tubular structure had an outer diameter of 3.6 mm, an inner diameter of 2 mm, and a length of 7.2 mm.

[Example 5] Production of cell structure (mosaic cell mass) using recombinant peptide block
NHBE (5000 cells / well), HUVEC (4000 cells / well), and HBdSMC (1000 cells / well) (as described in Comparative Example 1) CBE3 block prepared in Example 4 (53-106 μm) (7.5 μg / well) was suspended in 200 μl / well of a medium mixed with an equal amount of each cell's dedicated medium (described in Comparative Example 1) and seeded in each well of a PrimeSurface 96-well plate (Sumitomo Bakelite). . Next, the mixture of the cells and the CBE3 block was collected at the bottom of the well by centrifuging at 600 g for 5 minutes in a plate centrifuge, and then cultured at 37 ° C. for 3 days in a CO ₂ incubator.

In addition, the conditions for preparing the mosaic cell mass in a size that can be stacked were examined. Mosaic cell masses were prepared under a total of 9 conditions including the total number of cells (1 × 10 ⁴ , 1.5 × 10 ⁴ , or 2 × 10 ⁴ cells / well) and the amount of CBE3 block (3.75, 5.25, 7.5 μg / well). The results are shown in FIG. These were all sizes that could be stacked. In this example, with the intention of increasing the strength of the structure, conditions of a total cell number of 1 × 10 ⁴ cells / well and a CBE3 block amount of 7.5 μg / well were employed.

  The mosaic cell mass prepared by the above method is laminated on a support having the arrangement shown in FIG. 2 and FIG. 4 using a bio 3D printer “Regenova” (registered trademark) (Saifu Uses), and then the mosaic cell mass. Was cultured for 3 days using a medium having the same composition. In FIG. 4, the diameter of one needle-like body is 180 μm, and the interval between the needle-like bodies is 400 μm.

  In the tubular structure for tensile strength test, 48 needle-shaped bodies (reference numeral 32 in FIG. 4) of the ring-shaped needle-shaped bodies arranged in 9 × 9 are prepared for the production of one tubular structure. The mosaic cell mass was penetrated. For each needle-like body, 8 mosaic cell clusters (that is, 8 layers) were penetrated. That is, since one layer of the tubular structure is provided with eight layers of 48 mosaic cell clusters per layer, the total number of mosaic cell clusters is 384. The manufactured tubular structure had an outer diameter of 3.6 mm, an inner diameter of 2 mm, and a length of 3.2 mm.

  In the tubular structure for the water permeability test, for the production of one tubular structure, the ring-shaped 48 needle-shaped bodies (reference numeral 32 in FIG. 4) among the needle-shaped bodies arranged in 9 × 9. The mosaic cell mass was penetrated. In addition, for each needle-like body, 18 mosaic cell clusters (that is, 18 layers) were penetrated. That is, for each tubular structure, 18 layers comprising 48 mosaic cell masses are provided per layer, so the total number of mosaic cell masses is 864. The manufactured tubular structure had an outer diameter of 3.6 mm, an inner diameter of 2 mm, and a length of 7.2 mm.

  Thereafter, the tubular structure, which is a fusion of cell structures, was removed from each needle and then cultured for 3, 7, 14, 21, and 28 days.

[Example 6] Water permeability test The water permeability test was conducted with reference to 8.2.3 "Determination of integral water permeability / leakage" of ISO7198. In the water permeability test, a method was used in which the internal pressure of the flow path was increased in steps of 4, 8, 12, 16 kPa in steps of 30 seconds. In this process, the presence or absence of liquid leakage from the side wall of the structure or liquid leakage due to cracks in the structure was observed. Since living tissue is handled, PBS (phosphate buffered saline) was used as the solvent in the flow path instead of water. An overall view of the apparatus is shown in FIG. The pressure is reduced to 4, 8, 12, 16 kPa by the decompressor 2, and the pump is 0.1 MPa or more.

  Only the cells produced in Comparative Example 1 were subjected to the water permeability test described above using the spheroid-derived tubular structure and the mosaic cell mass-derived tubular structure produced in Example 5.

  As a result, as shown in FIG. 7 and FIG. 8, the tubular structure derived from spheroids only of cells does not show water permeability even when the water pressure is increased, and is derived from spheroids at the water pressure of 16 kPa, which has a breaking strength. The structure broke and water leaked vigorously. From this, it was shown that the spheroid-derived structure containing only cells cultured for 3 days has a breaking strength of 16 kPa and does not have water permeability unless it breaks.

  In the case of the tubular structure derived from the mosaic cell mass cultured in Example 5 and cultured for 3 days, the water permeability was exhibited even when the water pressure was 4 kPa. After that, even when the water pressure was increased, water permeability was confirmed, and when the water pressure was 16 kPa, the tubular structure derived from the mosaic cell mass was broken and water leaked vigorously. From this, the breaking strength of a tubular structure derived from a mosaic cell mass cultured for 3 days is 16 kPa, similar to a tubular structure derived from a cell-only spheroid, and what is a tubular structure derived from a cell-only spheroid? In contrast, it was found that even when it did not break, it showed high water permeability.

Example 7 Tensile Strength Test of Tubular Structure Derived from Mosaic Cell Mass A tensile strength test was performed according to ISO 7198 8.3.1 “Determination of cyclic tensile strength”. The apparatus used was Tissue Puller-560TP (DMT). A tubular structure was attached to this apparatus, and the maximum tensile load (mN) when the tubular structure was pulled in the circumferential direction at a constant speed was measured. Furthermore, the value obtained by dividing this value by the length of the tubular structure piece was shown as the tensile strength (mN / mm) per length of the tubular structure.

  Using the tubular structure produced in Example 5 after culturing for 3 days, 7 days, 14 days, 21 days, and 28 days after removing the tubular structure laminated and fused on the support from the support. The tensile strength test was performed as described above.

  Table 1 shows the maximum tensile load (mN) and tensile strength per length (mN / mm) of the tubular structure after culturing for 3 days, 7 days, 14 days, 21 days, and 28 days. The tensile strength per length (mN / mm) is also shown in FIG.

  From the above results, it can be seen that even a tubular structure cultured for 3 days has sufficient strength for practical use, but by culturing for a longer period of time, the tubular structure derived from the mosaic cell mass is improved in strength. It was. Compared with the tubular structure cultured for 3 days, the tubular structure cultured for 28 days was found to have 8 times or more strength.

21 Inner diameter 22 Outer diameter 23 Length 10 Support 11 Substrate 12 Filament or needle 1 Weigh scale 2 Depressurizer 3 Pump 4 Pressure gauge 5 Injection tube 6 Specimen 7 Plug 8 Bottle for pneumatic feeding 9 1% human serum Contains PBS (phosphate buffered saline)

Claims (16)

A cell structure comprising a biocompatible polymer block, fibroblasts, vascular endothelial cells and smooth muscle cells, wherein a plurality of the polymer blocks are arranged in a space between the plurality of cells. Tubular structure. The tubular structure according to claim 1, which is an artificial blood vessel. The said cell structure contains 0.0000001 microgram or more and 1 microgram or less bioaffinity polymer block per cell about the total cell number of a fibroblast, a vascular endothelial cell, and a smooth muscle cell of Claim 1 or 2 Tubular structure. The tubular structure according to any one of claims 1 to 3, wherein the fibroblasts are skin fibroblasts, the vascular endothelial cells are umbilical vein vascular endothelial cells, and the smooth muscle cells are bladder smooth muscle cells. . The tubular structure according to any one of claims 1 to 4, wherein a size of the biocompatible polymer block is 10 µm or more and 300 µm or less. The tubular structure according to any one of claims 1 to 5, wherein the tubular structure has an inner diameter of 1 mm to less than 6 mm, an outer diameter of 3 mm to 10 mm, and a length of 3 mm to 300 mm. The tubular structure according to any one of claims 1 to 6, wherein the biocompatible polymer block is composed of a recombinant peptide or a chemically synthesized peptide. The tubular structure according to any one of claims 1 to 7, wherein the recombinant peptide is recombinant gelatin or chemically synthesized gelatin. The tubular structure according to claim 8, wherein the recombinant gelatin or chemically synthesized gelatin is represented by the following formula 1.
Formula 1: A-[(Gly-XY) _n ] _m- B
In formula 1, each of n Xs independently represents any of amino acids, each of N Ys independently represents any of amino acids, m represents an integer of 2 to 10, and n represents 3 to 100 An integer is shown, A represents any amino acid or amino acid sequence, and B represents any amino acid or amino acid sequence. The recombinant peptide or chemically synthesized gelatin is
A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;
A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility; or 80% or more of the amino acid sequence described in SEQ ID NO: 1 A peptide having an amino acid sequence having the following sequence identity and having biocompatibility;
The tubular structure according to any one of claims 7 to 9, which is any one of the following. The tubular structure according to any one of claims 1 to 10, wherein in the biocompatible polymer block, the biocompatible polymer is crosslinked by heat, ultraviolet rays, or an enzyme. The tubular structure according to any one of claims 1 to 11, wherein the biocompatible polymer block is in the form of granules obtained by pulverizing a porous body of a biocompatible polymer. A method for producing a cell structure comprising a biocompatible polymer block and cells, wherein a plurality of the polymer blocks are disposed in a gap between the plurality of cells, and having an arbitrary shape,
A support having a substrate and a filamentous or needlelike body, the filamentous or needlelike body comprising a biocompatible polymer block and cells, wherein a plurality of the high heights are provided in the gaps between the plurality of cells. Step A in which a plurality of cell structures, in which molecular blocks are arranged, is penetrated, and the cell structure is arranged so that the arbitrary shape is formed by the plurality of cell structures, and the arrangement in Step A A step B of culturing the obtained cell structure and fusing adjacent cell structures together;
A method for producing a cell structure, comprising: Furthermore, the manufacturing method of the cell structure of Claim 13 including the process C which draws out the cell structure which formed arbitrary shapes in the process B from a filamentous body or a needle-shaped body. It is a manufacturing method of the tubular structure according to any one of claims 1 to 12,
A support having a substrate and a filamentous or needlelike body, the filamentous or needlelike body comprising a biocompatible polymer block and cells, wherein a plurality of the high heights are provided in the gaps between the plurality of cells. Step A1 in which a plurality of cell structures in which molecular blocks are arranged are penetrated, and the cell structure is arranged so that a shape of a tubular structure is formed by the plurality of cell structures, wherein the cells Step B1, comprising fibroblasts, vascular endothelial cells and smooth muscle cells, and culturing the cell structure disposed in step A1 to fuse adjacent cell structures;
A method for manufacturing a tubular structure, comprising: The step C1 further includes a step C1 of pulling out the cell structure formed in the shape of the tubular structure in the step B1 from the filamentous body or needle-like body, and a step D1 of culturing the cell structure obtained in the step C1. The manufacturing method of the tubular structure of description.