JP2015134193A - Cell structure for cell transplantation, and cell aggregate for cell transplantation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell structure for cell transplantation, capable of having a thickness suitable for cell transplantation, preventing the necrosis of transplanted cells, and forming vessels at a transplantation site after transplantation.SOLUTION: The cell structure for cell transplantation includes polymer blocks having biocompatibility and at least one kind of cells. A plurality of polymer blocks are disposed in the gap between a plurality of cells.

Description

  The present invention relates to a cell structure for cell transplantation and a cell assembly for cell transplantation, and more specifically, a cell structure for cell transplantation that suppresses necrosis of the transplanted cells after transplantation and can form blood vessels at the transplantation site, The present invention also relates to a cell assembly for cell transplantation capable of forming blood vessels at a transplant site.

  Currently, regenerative medicine is being put to practical use that measures the regeneration of biological tissues and organs that have fallen into dysfunction or dysfunction. Regenerative medicine is a new form that regenerates the same form and function as the original tissue by using the three factors of cells, scaffolding, and growth factors. Medical technology. In recent years, treatment using cells has been gradually realized. For example, cultured epidermis using autologous cells, cartilage treatment using autologous chondrocytes, bone regeneration treatment using mesenchymal stem cells, cardiomyocyte sheet treatment using myoblasts, cornea regeneration treatment using corneal epithelial sheet, Examples include nerve regeneration treatment. Unlike these alternative medicines (artificial bone filling agents and hyaluronic acid injection), these new treatments can restore and regenerate living tissue, and thus can achieve high therapeutic effects. In fact, products such as cultured epidermis and cultured cartilage using autologous cells have been put on the market.

  Here, for example, in the regeneration of myocardium using a cell sheet, it is considered that a multilayer structure of cell sheets is necessary to regenerate a thick tissue. Recently, Okano et al. Developed a cell sheet using a temperature-responsive culture dish. Since the cell sheet does not require an enzyme treatment such as trypsin, the cell-to-cell bond and the adhesion protein are retained. (Non-Patent Documents 1 to 6). Such a cell sheet manufacturing technique is expected to be useful for regeneration of myocardial tissue (Non-patent Document 7). Okano et al. Believe that it is impossible to achieve a thickness of 200 μm or more, and are developing a cell sheet in which vascular endothelial cells are introduced together to form a vascular network in the cell sheet (non-patent document). 8).

In bone regeneration, a bone regeneration sheet in which cultured cells are added to a base material has been developed.
A bone regeneration sheet (Patent Document 1) is proposed in which a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet form and a biodegradable sheet obtained by forming a biodegradable substance in a sheet form are laminated. There is also a mesenchymal tissue regeneration-inducing sheet in which mesenchymal tissue precursor cells differentiated from mesenchymal cells and an extracellular matrix are attached on a porous sheet (Patent Document 2). After that, in Patent Document 3, it is stated that a sheet having a thickness of 200 μm or more can be formed by development and optimization of the culture technique, but it has been confirmed that a cortical bone tissue layer of about 210 μm has been formed. Yes.

  Furthermore, as a means for solving the fact that nutrients do not sufficiently permeate into a three-dimensional structure composed of cells only by diffusion, a gel-embedded culture using collagen has been devised (Non-patent Document 9). ). Patent Document 4 states that three-dimensional culture is possible by connecting cells with inorganic ceramic beads.

JP 2003-275294 A JP 2006-116212 A JP 2009-240766 A JP 2004-267562 A

Shimizu, T. et al., Circ. Res. 90, e40-48 (2002) Kushida, A. et al., J. Biomed. Mater. Res. 51, 216-223 (2000) Kushida, A. et al., J. Biomed. Mater. Res. 45, 355-362 (1999) Shimizu, T., Yamato, M., Kikuchi, A. & Okano, T., Tissue Eng. 7, 141-151 (2001) Shimizu, T et al., J. Biomed. Mater. Res. 60,110-117 (2002) Harimoto, M. et al., J. Biomed. Mater. Res. 62, 464-470 (2002) Shimizu, T., Yamato, M., Kikuchi, A. & Okano, T., Biomaterials 24, 2309-2316 (2003) Inflammation and Regeneration vol.25 No.3 2005, p158-159. The 26th Annual Meeting of Japanese Inflammation and Regenerative Medicine-Aiming at Fusion of Inflammation and Regenerative Medicine-Mitsuo Okano Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.J Yang, J Richards, P Bowman, R Guzman, J Enami, K McCormick, S Hamamoto, D Pitelka, and S Nandi.PNAS July 1, 1979 vol. 76 no. 7 3401-3405

  In the current technology relating to regenerative medicine, cells to be transplanted are mainly transplanted in a thin sheet form or transplanted in a suspension state, and thus a tissue having a sufficient thickness cannot be provided. The living tissue originally has a thickness, and thus has a thickness. Therefore, the living tissue enables a muscular force that beats the heart, and enables a smooth movement in the articular cartilage. In general tissue regeneration using cells, it is considered that a major problem is that a tissue having a thickness cannot be provided.

  Conventional cell sheets cannot form a vascular network, and thus it is difficult to regenerate a sufficiently thick tissue (Non-patent Documents 5 and 7). The reason is that by providing the cell sheet with a thickness, the nutrient supply to the cells in the center is interrupted and the cells die. In addition, a cell sheet in which vascular endothelial cells are introduced together (Non-Patent Document 8) has been developed. This requires that, in addition to the target cells, another cell source called vascular endothelial cells must be prepared. Even if it is difficult to induce blood vessels uniformly in the cell sheet, and even if this method can provide a nutrient delivery route, this method can be used to make the created nutrient delivery route more precise than the external nutrient delivery route. It has a lot of problems, such as having to be combined, and is not a realistic solution.

  The inventions described in Patent Documents 1 and 2 described above are methods in which a sheet with cultured osteoblasts attached is placed in the body, and cortical bone is formed from osteoblasts by membrane ossification in the body. However, since osteoblast-like cells cannot be laminated and cultured, a sheet using an osteoblast layer could not provide a regenerated sheet having a cell layer thickness exceeding 100 μm.

  As described above, conventionally, it has been difficult to provide cells as a thick composition in many tissue repairs. The main cause is that nutrients do not sufficiently penetrate into a three-dimensional structure composed of cells only by diffusion. As a means for solving this problem, a gel-embedded culture using collagen has been devised (Non-Patent Document 9). However, in cells embedded in a gel, the cells move from the center of the gel to the outside, so the cells do not exist uniformly in the gel and the cell density in the center is low, which is fundamentally This problem cannot be solved. In addition, in a cell three-dimensional structure produced by gel embedding, the three-dimensional structures cannot be joined and fused together, and a three-dimensional structure larger than the size produced at the time of cell seeding cannot be formed. Therefore, it is not possible to take a means of producing a structure in which cells are uniformly distributed by producing a small gel and then fusing the gels together.

  As described above, Patent Document 4 states that three-dimensional culture is possible by connecting cells with inorganic ceramic beads. However, inorganic ceramics have poor water retention, liquid exchange, nutrient diffusion, and buffering ability, and have not been able to provide a cell composition with a realistic thickness. In fact, even in the example of Patent Document 4, cells are adhered to particles of 150 to 460 μm, and a thick non-woven fabric (1 cm) of PLLA is laminated around them to increase the apparent thickness. The inclusion layer is only a few tens of μm layer on the surface of the inorganic ceramic beads. Even if a 1 cm PLLA non-woven fabric having no cells is regarded as a structure, it is only a structure having a remarkably uneven cell distribution. Thus, until now, it has only been possible to provide a three-dimensional cell structure with a non-uniform cell distribution in the structure and a structure with a thin substantial cell layer.

As described above, the conventional technology does not provide a sufficient thickness suitable for cell transplantation, suppression of necrosis of transplanted cells, and sufficient biomaterials for angiogenesis. A biomaterial for cell transplantation that satisfies the above has been desired.
Therefore, the present invention can have a thickness suitable for cell transplantation, suppresses necrosis of the transplanted cells, and can form a blood vessel at the transplant site after transplantation. An object was to provide a structure.

  As a result of intensive studies to solve the above problems, the inventors of the present invention include a biocompatible polymer block (containing a biocompatible polymer material) as a cell structure for cell transplantation used for cell transplantation. The inventors have found that the above-described object can be achieved by using a material having a specific arrangement of lumps and cells, and have completed the present invention.

That is, the cell structure for cell transplantation of the present invention comprises a polymer block having biocompatibility and at least one kind of cell, and a plurality of the polymer blocks are provided in the gaps between the plurality of cells. It is characterized by being arranged.
In the present invention, the size of the polymer block is preferably 1 μm or more and 700 μm or less, and more preferably 10 μm or more and 300 μm or less. Moreover, the cell structure for cell transplantation of the present invention preferably has a thickness or diameter of 400 μm or more and 3 cm or less, and preferably 720 μm or more and 1 cm or less. Further, the ratio of the polymer block to the cells is preferably 0.0000001 μg or more and 1 μg or less per cell. Furthermore, it is preferably produced by incubating a mixture of a biocompatible polymer block and a cell-containing culture solution.

Preferably, the biocompatible polymer is a biodegradable material, polypeptide, polylactic acid, polyglycolic acid, PLGA, hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, Examples are chitin or chitosan, preferably gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, or retronectin.
Preferably, the biocompatible polymer is crosslinked, and more preferably, the crosslinking is performed with aldehydes, a condensing agent, or an enzyme.

Preferably, the polymer having bioaffinity is a recombinant peptide and has two or more cell adhesion signals in one molecule. Preferably, the recombinant peptide is
Formula: A-[(Gly-XY) _n ] _m -B
(In the formula, A represents an arbitrary amino acid or amino acid sequence, B represents an arbitrary amino acid or amino acid sequence, n Xs independently represent any of the amino acids, and n Ys each independently represent an amino acid. N represents an integer of 3 to 100, and m represents an integer of 2 to 10. Note that n Gly-XY may be the same or different. Yes, more preferably
Formula: Gly-Ala-Pro-[(Gly-XY) ₆₃ ] ₃ -Gly
(In the formula, 63 X's each independently represent any amino acid, and 63 Y's each independently represent any amino acid. The 63 Gly-XYs may be the same or different. Good.) Preferably, the recombinant peptide is (1) an amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence having bioaffinity having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1. It has an arrangement. The cell structure for cell transplantation of the present invention preferably contains an angiogenic factor.

The cell according to the present invention is preferably a cell selected from the group consisting of a universal cell, somatic stem cell, progenitor cell and mature cell, and a non-vascular cell is used as the cell structure for cell transplantation of the present invention. What is contained can also be used suitably and the aspect whose cell is only a non-vascular cell can also be used suitably. It is preferable that the number of the cells is two or more and includes both a non-vascular cell and a vascular cell. In this case, the area of the central vascular cell in the cell structure is equal to the peripheral vascular system. It is more preferable to have a region larger than the area of the cells, and it is more preferable that the ratio of the vascular cells in the central part is 60% to 100% with respect to the total area of the vascular cells. Moreover, it is preferable that the cell structure has a region where the cell density of the vascular system at the center is 1.0 × 10 ⁻⁴ cells / μm ³ or more. In the cell transplant cell structure of the present invention, the cell transplant cell structure of the present invention includes two or more types of cells and includes both non-vascular cells and vascular cells. Including those formed.

Moreover, the cell aggregate for cell transplantation of the present invention is a cell aggregate for cell transplantation composed of non-vascular cells and vascular cells,
(1) the area of the vascular system cells in the center of the cell assembly has a region larger than the area of the peripheral vascular cells; and (2) the cell density of the vascular system in the center is 1. Having a region of 0 × 10 ⁻⁴ cells / μm ³ or more,
Is characterized by satisfying at least one of the requirements.
The cell aggregate for cell transplantation of the present invention preferably satisfies both the above requirements (1) and (2), and the ratio of the cells in the central vasculature is relative to the total area of vascular cells. It is also preferable to have a region that is 60% to 100%.

  The cell structure for cell transplantation of the present invention can have a thickness suitable for cell transplantation and contains a biocompatible polymer block (containing a biocompatible polymer material). The cells and the cells are arranged three-dimensionally in a mosaic pattern to form a three-dimensional cell structure in which the cells are uniformly present in the structure, and to deliver nutrients from the outside to the inside of the cell three-dimensional structure. It becomes possible. Thereby, when cell transplantation is performed using the cell structure for cell transplantation of the present invention, necrosis of the transplanted cells is suppressed and transplantation becomes possible. Furthermore, even when vascular cells are not used as the cell type to be used, blood vessels can be formed at the transplant site after transplantation. In addition, the cell aggregate for cell transplantation of the present invention can form blood vessels at the transplantation site after transplantation.

FIG. 1 shows a stereoscopic microscope photograph of Day 7 (cartilage differentiation medium) of a mosaic cell mass prepared with a recombinant peptide μ block. FIG. 2 shows a stereomicrograph of Day 7 (cartilage differentiation medium) of a mosaic cell mass prepared with a natural gelatin μ block. FIG. 3 shows a photograph of a section of a mosaic cell mass using a recombinant peptide μ block (HE staining × 5 times). FIG. 4 shows a photograph of a section of a mosaic cell mass using a recombinant peptide μ block (HE staining × 10 times). FIG. 5 shows a photograph of a section of a mosaic cell mass using a recombinant peptide μ block (HE staining × 40 times). FIG. 6 shows the fusion of the mosaic cell mass. FIG. 7 shows a HE-stained section photograph (× 5 times) of fusion of mosaic cell masses (fusion of 3 mosaic cell masses). FIG. 8 shows a HE-stained section photograph (× 10 times) of fusion of mosaic cell masses (fusion of 3 mosaic cell masses). FIG. 9 shows a HE-stained section photograph (× 20 times) of fusion of mosaic cell masses (fusion of 3 mosaic cell masses). FIG. 10 shows a HE-stained section photograph (× 5) of fusion of mosaic cell masses (fusion of 3 mosaic cell masses). FIG. 11 shows a HE-stained section photograph (× 10 times) of fusion of mosaic cell masses (fusion of 3 mosaic cell masses). FIG. 12 shows a stereoscopic microscope photograph (change over time) of the volume of the mosaic cell mass increased. FIG. 13 shows the time-dependent change in diameter from a stereomicrograph of a mosaic cell mass with increased volume. FIG. 14 shows the time-dependent change of the area from the stereomicrograph of the mosaic cell mass which increased in volume. FIG. 15 shows the change over time of the volume (4 / 3πr ³ ) calculated by calculation from a stereomicrograph of the mosaic cell mass whose volume has been increased. FIG. 16 shows a section of a mosaic cell mass (Day 7 (under growth medium), × 5) using a recombinant peptide μ block. FIG. 17 shows a section of a mosaic cell mass (Day 7 (under growth medium), × 10 times) using the recombinant peptide μ block. FIG. 18 shows a HE section photograph (× 5 times) of Day 21 (with a recombinant peptide block added under growth medium) whose volume has been increased. FIG. 19 shows Day21 with increased volume (recombinant peptide block added in growth medium) HE section photograph (× 40 times) FIG. 20 shows HE section photographs (× 5 times, × 20 times) of Day 21 (with a recombinant peptide block added under cartilage differentiation medium) whose volume was increased. FIG. 21 shows GAG spectrum data. FIG. 22 shows the change over time in the amount of GAG production in the mosaic cell mass. FIG. 23 shows the amount of ATP produced and retained by the cells in the mosaic cell mass (Day 7). FIG. 24 shows a stereoscopic microscope photograph of Day2 (growth medium) of a mosaic cell mass prepared with a PLGAμ block. FIG. 25 shows that the mosaic cell mass using cardiomyocytes and the recombinant peptide μ block is pulsating synchronously as a whole. FIG. 26 shows a fluorescence micrograph and a micrograph of a mosaic cell mass using GFP-expressing HUVEC and recombinant peptide μ block. (50,000 cells + 0.03 mg mosaic cell mass and 300,000 cells + 0.2 mg mosaic cell mass) FIG. 27 shows a stereomicrograph of a fused mosaic cell mass. FIG. 28 shows a cross-sectional photograph (HE staining) of a mosaic cell mass using the recombinant peptide μ block. FIG. 29 shows a cross-sectional photograph (HE staining) of a cell mass of hMSC. FIG. 30 shows a cross-sectional photograph (immunostaining using CD29 antibody and CD31 antibody) of a mosaic cell mass produced in Example 20- (1) using the recombinant peptide. FIG. 31 shows a cross-sectional photograph (immunostaining using CD29 antibody and CD31 antibody) of a mosaic cell mass produced in Example 20- (2) A and using a recombinant peptide. FIG. 32 shows a cross-sectional photograph (immuno-staining using CD29 antibody and CD31 antibody) of a mosaic cell mass using the recombinant peptide produced in Example 20- (2) B. FIG. 33 shows a photograph of a section of a mosaic cell mass produced in Example 20- (3) using a recombinant peptide (immunostaining using CD29 antibody and CD31 antibody). FIG. 34 shows a tissue section photograph (HE staining) of the transplanted site after transplantation of the mosaic cell mass (Example 19- (1)) using the recombinant peptide μ block. FIG. 35 shows a tissue section photograph (HE staining) of the transplanted site after transplantation of hMSC cell mass (Comparative Example 1). FIG. 36 shows a tissue section photograph (HE staining) of the transplanted site after transplantation of the mosaic cell mass (Example 20- (1)) using the recombinant peptide μ block. FIG. 37 shows a tissue section photograph (HE staining) of a transplanted site after transplantation of a mosaic cell mass (Example 20- (2)) A using a recombinant peptide μ block. FIG. 38 shows a tissue section photograph (HE staining) of the transplanted site after transplantation of the mosaic cell mass (Example 20- (3)) using the recombinant peptide μ block. FIG. 39 shows a tissue section photograph (HE staining) of a transplanted site after transplantation of a mosaic cell mass (Example 20- (2) A) using a recombinant peptide μ block. FIG. 40 shows a tissue section photograph (HE staining) of a transplanted site after transplantation of a mosaic cell mass (Example 20- (2) B) using a recombinant peptide μ block. FIG. 41 shows a tissue section photograph (HE staining) of the transplanted site after transplanting the cell mass (B of Comparative Example 2).

Hereinafter, embodiments of the present invention will be described in detail.
The cell structure for cell transplantation of the present invention comprises a polymer block having biocompatibility and at least one kind of cell, and a plurality of the polymer blocks are arranged in a gap between the plurality of cells. It is characterized by this. As an embodiment, one or more of a plurality of polymer blocks having biocompatibility and a plurality of cells, wherein one or more of the plurality of gaps formed by the plurality of cells are part or all of Examples thereof include a cell structure in which the polymer block is disposed.

  Although the shape of the polymer block according to the present invention is not particularly limited, for example, it is indefinite, spherical, particulate, powdery, porous, fibrous, spindle-shaped, flat-shaped and sheet-shaped, Preferably, they are amorphous, spherical, particulate, powdery and porous, more preferably amorphous. An indeterminate shape indicates that the surface shape is not uniform, for example, an object having irregularities such as rocks.

  In the cell structure for cell transplantation of the present invention, a plurality of the polymer blocks are arranged in the gaps between the plurality of cells. 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 cells via the polymer block, that is, the gap distance when selecting a cell and a cell that is the shortest distance from the cell is not particularly limited, but the size of the polymer block The preferred distance is also within the preferred size range of the polymer block.

The polymer block according to the present invention is sandwiched between cells, but there is no need for cells between all the polymer blocks, and there may be locations where the polymer blocks are in contact with each other. . The distance between the polymer blocks through the cells, that is, the distance when selecting the polymer block and the polymer block present at the shortest distance from the polymer block is not particularly limited, but the cell used Is preferably the size of a cell mass when 1 to several cells are collected, for example, 10 μm or more and 1000 μm or less, preferably 10 μm or more and 100 μm or less, and more preferably 10 μm or more and 50 μm or less.
In this specification, the expression “is uniformly present” such as “a three-dimensional structure of cells in which cells uniformly exist in the structure” is used, but it does not mean complete uniformity. In addition, it is possible to deliver nutrients from the outside to the inside of the three-dimensional cell structure, which is the effect of the present invention, to prevent necrosis of the transplanted cells, and after transplantation, blood vessels It means that it is distributed in a range where it can be formed.

(1) Polymer material having biocompatibility (1-1) Polymer material If the polymer having bioaffinity used in the present invention has affinity for a living body, is it decomposed in vivo? Although it is not specifically limited whether it is comprised with a biodegradable material. Specifically, the non-biodegradable material is at least selected from the group consisting of PTFE, polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, stainless steel, titanium, silicone, and MPC (2-methacryloyloxyethyl phosphorylcholine). One material. Specific examples of biodegradable materials include polypeptides, polylactic acid, polyglycolic acid, PLGA (polylactic acid-co-glycolic acid), hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose. At least one material selected from the group of chitin and chitosan. Of the above, polypeptides are particularly preferred. These polymer materials may be devised to enhance cell adhesion. Specific methods are as follows. “Cell adhesion substrate (fibronectin, vitronectin, laminin) and cell adhesion sequence (expressed by one letter code of amino acid, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence) coating with peptide ", 2. 2. “Amination and cationization of substrate surface”; Methods such as “plasma treatment of substrate surface, hydrophilic treatment by corona discharge” can be used.

  The type of polypeptide is not particularly limited as long as it has biocompatibility.For example, gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, and retronectin are most preferable. Gelatin, collagen and atelocollagen are preferred. As gelatin for use in the present invention, natural gelatin or a recombinant peptide is preferable. More preferably, it is a recombinant peptide. As used herein, natural gelatin means gelatin made from naturally derived collagen. The recombinant peptide will be described later in this specification.

The hydrophilic 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 / nonpolarity of an organic compound proposed by Satoshi Fujita, and details thereof are described 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 (CH4) is the source of all organic compounds, and all other compounds are all methane derivatives, with certain numbers set for their carbon number, substituents, transformations, rings, etc. 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. Is. The IOB in the organic conceptual diagram refers to 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 it contributes to the stabilization and survival of cells in the three-dimensional cell structure (mosaic cell mass) of the invention.

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 0.3 or less and minus 9.0 or more. Preferably, it is 0.0 or less and more preferably 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 in the above range, it has high hydrophilicity and high water absorption, so that it effectively acts on retention of nutrient components, and as a result, the cell 3 of the present invention. It is presumed that it contributes to the stabilization and survival of cells in a dimensional structure (mosaic cell mass; mosaic cell mass).

(1-2) Crosslinking The polymer material having bioaffinity used in the present invention may be crosslinked or uncrosslinked, but is preferably crosslinked. Crosslinking methods include thermal crosslinking, chemical crosslinking, crosslinking with aldehydes (eg, formaldehyde, glutaraldehyde, etc.), crosslinking with condensing agents (carbodiimide, cyanamide, etc.), enzyme crosslinking, photocrosslinking, UV crosslinking, hydrophobic interaction, Although known methods such as hydrogen bonding and ionic interaction can be used, a crosslinking method using glutaraldehyde and a thermal crosslinking method are preferred.

  Examples of photocrosslinking include photoirradiation to a polymer into which a photoreactive group has been introduced, or light irradiation in the presence of a photosensitizer. Examples of the photoreactive group include a cinnamyl group, a coumarin group, a dithiocarbamyl group, a xanthene dye, and camphorquinone.

  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. Preferably, trans-glutaminase and laccase, most preferably trans-glutaminase can be used for cross-linking. . A specific example of a protein that is enzymatically cross-linked with transglutaminase is not particularly limited as long as it has 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 crosslinking of the polymer material has two processes: a process of mixing a solution of the polymer material and a crosslinking agent and a process of reacting these solutions.

  In the present invention, the mixing temperature when the polymer material is treated with the crosslinking agent is not particularly limited as long as the solution can be mixed, but is preferably 0 ° C to 100 ° C, more preferably 0 ° C to 40 ° C, More preferably, it is 0 degreeC-30 degreeC, More preferably, it is 3 degreeC-25 degreeC, More preferably, it is 3 degreeC-15 degreeC, More preferably, it is 3 degreeC-10 degreeC, Most preferably, it is 3 degreeC- 7 ° C.

  In the reaction process of the polymer material and the crosslinking agent, the temperature can be increased. The reaction temperature is not particularly limited as long as the crosslinking proceeds, but is substantially -100 ° C to 200 ° C, more preferably 0 ° C to 60 ° C in consideration of modification and decomposition of the polymer material. More preferably, it is 0 degreeC-40 degreeC, More preferably, it is 3 degreeC-25 degreeC, More preferably, it is 3 degreeC-15 degreeC, More preferably, it is 3 degreeC-10 degreeC, Most preferably, it is 3 degreeC- 7 ° C.

  Further, the polymer material can be crosslinked without using a crosslinking agent. The crosslinking method is not particularly limited, and specific examples include a thermal crosslinking method.

  The reaction temperature at the time of crosslinking without using a crosslinking agent 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 50 ° C to 300 ° C, more preferably 100 ° C to 250 ° C, and still more preferably 120 ° C to 200 ° C.

(1-3) Recombinant peptide The recombinant peptide according to the present invention means a polypeptide or protein-like substance having a gelatin-like amino acid sequence produced by a gene recombination technique. The recombinant peptide that can be used in the present invention preferably has a repeating sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents an amino acid) (a plurality of Gly -XY may be the same or different. Preferably, two or more sequences of cell adhesion signals are contained in one molecule. As the recombinant peptide used in the present invention, a recombinant peptide having an amino acid sequence derived from a partial amino acid sequence of collagen can be used. For example, those described in EP1014176, US6992172, WO2004 / 85473, WO2008 / 103041, etc. can be used. However, it is not limited to these. What is preferable as a recombinant peptide used by this invention is the recombinant peptide of the following aspects.

  The recombinant peptide used in the present invention is excellent in biocompatibility due to the inherent properties of natural gelatin, and is not naturally derived. In addition, since the recombinant peptide used in the present invention is more uniform than the natural peptide and the sequence is determined, the strength and degradability can be precisely designed with less blur due to the crosslinking described later. is there.

  The molecular weight of the recombinant peptide is preferably 2 KDa or more and 100 KDa or less. More preferably, it is 2.5 KDa or more and 95 KDa or less. More preferably, it is 5 KDa or more and 90 KDa or less. Most preferably, it is 10 KDa or more and 90 KDa or less.

  The recombinant peptide has a repeating sequence represented by Gly-XY characteristic of collagen. Here, the plurality of Gly-X-Ys may be the same or different. In Gly-X—Y, Gly represents glycine, and X and Y represent any amino acid (preferably any amino acid other than glycine). The GXY sequence 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 of the sequence, more preferably 95% or more, and most preferably 99% or more of the amino acids are GXY repeating structures.

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

  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 peptide 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 sequence, LRE sequence, DGEA sequence, and HAV sequence are preferable, RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence, and HAV sequence, and particularly preferably RGD sequence. Of the RGD sequences, an ERGD sequence is preferred. By using a recombinant peptide 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.

  As the arrangement of the RGD sequence in the recombinant peptide used in the present invention, it is preferable that the number of amino acids between RGD 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 peptide 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 peptide contains 350 or more amino acids, each stretch of 350 amino acids has at least one RGD. It is preferable to include a 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 peptides of the invention comprise at least 2 RGD sequences per 250 amino acids, more preferably at least 3 RGD sequences per 250 amino acids, and even more preferably at least 4 per 250 amino acids. Contains the RGD sequence. As a further aspect of the recombinant peptide 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.

  Further, the recombinant peptide may be partially hydrolyzed.

  The recombinant peptide used in the present invention preferably has a repeating structure of A [(Gly-XY) n] mB. 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.

  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, and type V. More preferred are type I, type II and type III. According to another form, the collagen origin is preferably human, cow, pig, mouse, rat. More preferably, it is a human.

  The isoelectric point of the recombinant peptide 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 peptide is not deaminated.
Preferably, the recombinant peptide does not have a telopeptide.
Preferably, the recombinant peptide is a substantially pure collagen material prepared with a nucleic acid encoding natural collagen.

Particularly preferably as a recombinant peptide used in the present invention,
(1) the amino acid sequence of SEQ ID NO: 1; or (2) 80% or more (more preferably 90% or more, most preferably 95% or more) of homology with the amino acid sequence of SEQ ID NO: 1, An amino acid sequence having biocompatibility;
It is a recombinant peptide having

  The recombinant peptide used in the present invention can be produced by a gene recombination technique known to those skilled in the art. For example, it can be produced according to the method described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041, and the like. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant peptide 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 peptide is produced by culturing the obtained transformant in an appropriate medium. Therefore, the recombinant peptide used in the present invention can be prepared by recovering the recombinant peptide produced from the culture. .

(1-4) Polymer block having biocompatibility In the present invention, a lump containing the above-described polymer material having biocompatibility is used. The production method of the polymer block is not particularly limited. For example, a solid block made of a polymer is pulverized using a pulverizer (new power mill, etc.), and then a block having a desired size is obtained by sizing the sieve. can do.

The size of the polymer block is preferably 1 μm or more and 700 μm or less, more preferably 10 μm or more and 700 μm or less, further preferably 10 μm or more and 300 μm or less, further preferably 20 μm or more and 150 μm or less, and particularly preferably It is 25 μm or more and 106 μm or less. In addition, the polymer block may be in the form of a long string of 700 μm or more with the above-mentioned size as a thickness, and further may be in the form of a sheet or gel with the above-mentioned size as a thickness. By setting it as the said suitable range, a cell can exist more uniformly in a structure.
Moreover, it is preferable that the cell structure for cell transplantation of the present invention contains 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 for cell transplantation containing an angiogenic factor is not particularly limited, for example, it can be manufactured by using a polymer block impregnated with an angiogenic factor.

(2) Cell The cell used in the present invention can be appropriately used as long as it can perform cell transplantation, which is the object of the cell structure of the present invention, and the type thereof is not particularly limited. In addition, one type of cell may be used or a combination of a plurality of types may be used. In addition, the cells to be used are preferably animal cells, more preferably vertebrate cells, and particularly preferably human cells. Vertebrate-derived cells (particularly human-derived cells) may be any of universal cells, somatic stem cells, progenitor cells, or mature cells. For example, ES cells, GS cells, or iPS cells can be used as the universal cells. As the somatic stem cells, for example, mesenchymal stem cells (MSC), hematopoietic stem cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, myocardial stem cells, adipose-derived stem cells, or neural stem cells can be used. Examples of progenitor cells and mature cells include skin, dermis, epidermis, muscle, myocardium, nerve, bone, cartilage, endothelium, brain, epithelium, heart, kidney, liver, pancreas, spleen, oral cavity, cornea, bone marrow, umbilical cord Cells derived from blood, amniotic membrane, or hair can be used. Examples of human-derived cells include ES cells, iPS cells, MSCs, chondrocytes, osteoblasts, osteoprogenitor cells, mesenchymal cells, myoblasts, cardiomyocytes, cardioblasts, neurons, hepatocytes, Beta cells, fibroblasts, corneal endothelial cells, vascular endothelial cells, corneal epithelial cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, or hematopoietic stem cells can be used. In addition, the origin of the cell may be either an autologous cell or an allogeneic cell.

  For example, in heart diseases such as severe heart failure and severe myocardial infarction, myocardial cells, smooth muscle cells, fibroblasts, skeletal muscle-derived cells (especially satellite cells), bone marrow cells (especially myocardial cells) Preferred examples include bone marrow cells differentiated into). Furthermore, transplanted cells can be appropriately selected in other organs. For example, transplantation of neural progenitor cells or cells that can differentiate into nerve cells to cerebral ischemia / cerebral infarction, vascular endothelial cells or cells that can differentiate into vascular endothelial cells to myocardial infarction / skeletal muscle ischemia Implantation can be suitably performed.

  Moreover, the cell used for the cell transplant with respect to a diabetic organ disorder is mentioned. For example, cells for cell transplantation treatment that have been variously studied for diseases such as kidney, pancreas, peripheral nerves, eyes, and blood circulation disorders of the extremities. That is, attempts have been made to transplant insulin-secreting cells into the pancreas whose insulin secreting ability has been reduced, transplantation of bone marrow-derived cells for limb circulation disorders, and such cells can be used.

  As the cell structure for cell transplantation of the present invention, those containing non-vascular cells can be preferably used. In addition, the cells constituting the cell structure for cell transplantation of the present invention can be preferably used only with non-vascular cells, and only non-vascular cells can be used for cell transplantation of the present invention. With the cell structure, blood vessels can be formed at the transplant site after transplantation. Furthermore, when the cell structure for cell transplantation of the present invention comprises two or more types of cells and includes both non-vascular cells and vascular cells, the cell structure is composed of only non-vascular cells. Compared with the case where it exists, it becomes possible to form blood vessels more and is more preferable.

Furthermore, there are two or more types of cells constituting the cell structure for cell transplantation of the present invention, and the area of the vascular cells in the central part of the cell structure is larger than the area of the peripheral vascular cells. It is further preferable that blood vessels can be formed. Here, specifically, that the area of the cells of the vascular system in the central part of the cell structure is larger than the area of the cells of the vascular system in the peripheral part, specifically, an arbitrary 2 μm sliced specimen is obtained. It means that there is a specimen having the above region when manufactured. Here, the central part of the cell structure refers to an area having a distance of 80% from the center of the distance from the center to the surface of the cell structure, and the peripheral part of the cell structure is 80% from the center. % Area to the surface of the structure. The central part of the cell structure is determined as follows.
In an arbitrary cross-section passing through the center of the cell structure, the center of the circle with the radius X is moved along the extension of the cross-section, and the area of the portion excluding the overlapped portion of the moved circle and the cross-section is The radius X is calculated so that it is 64% of the area. The center of the circle with the radius X is moved once, and the portion excluding the overlapped portion of the moved circle and the cross section is taken as the center of the cell structure. At this time, the cross section having the largest cross-sectional area is most preferable. Note that the center of the cell structure is a portion of the cross section having the maximum cross-sectional area, in which the center of the circle with the radius Y is moved around the extension of the cross section and the overlapped portion of the moved circle and the cross section is excluded. The radius Y determined to be a single point is obtained. The center of the circle with the radius Y is moved once, and one point excluding the overlapped portion between the moved circle and the cross section is defined as the center of the cell structure. When a line segment is formed without being determined as one point, or when there are a plurality of line segments, each line segment is centered on a point that bisects its length.

  Specifically, it is preferable that the ratio of the vascular cells in the central part is 60% to 100% with respect to the total area of vascular cells, more preferably 65% to 100%, 80 % To 100% is more preferable, and 90% to 100% is more preferable. It should be noted that the fact that the ratio of the vascular cells in the central part is 60% to 100% with respect to the total area of the vascular cells specifically means that an arbitrary 2 μm slice When a specimen is prepared, it means that there is a specimen having the ratio area. By setting it as the said range, angiogenesis can be promoted more.

  The ratio of the vascular cells in the central part is determined by, for example, staining the vascular cells to be measured when a sliced sample is prepared and using the image processing software ImageJ, The average value of (intensity) is calculated and the area of the central portion × intensity is calculated. Further, the average value of the intensity (intensity) of the entire color is calculated, and the total area × intensity is calculated. It can also be calculated by determining the ratio of the area of the central portion × the intensity. Here, as a method of staining vascular cells, a known staining method can be appropriately used. For example, when hECFC is used as a cell, a CD31 antibody can be used.

Moreover, it is preferable that the cell structure has a region where the cell density of the vascular system in the central part is 1.0 × 10 ⁻⁴ cells / μm ³ or more. It is more preferable. Here, having a region that is 1.0 × 10 ⁻⁴ cells / μm ³ or more specifically refers to a sample having a region of the density when an arbitrary 2 μm sliced sample is prepared. Say that exists. The cell density is more preferably 1.0 × 10 ^{−4 to} 1.0 × 10 ⁻³ cells / μm ³ , further preferably 1.0 × 10 ^{−4 to} 2.0 × 10 ⁻⁴ cells / μm ^3. More preferably, it is 1.1 × 10 ^{−4 to} 1.8 × 10 ⁻⁴ cells / μm ³ , and further preferably 1.4 × 10 ^{−4 to} 1.8 × 10 ⁻⁴ cells / μm ³ . By setting it as the said range, angiogenesis can be promoted more.

  Here, the cell density of the vascular system at the center can be obtained by actually counting the number of cells in the sliced specimen and dividing the number of cells by the volume. Here, the central portion is determined as follows. A portion obtained by cutting out the thickness of the thin sliced sample in a direction perpendicular to the above-described central portion. The cell density can be determined by, for example, superimposing a sliced specimen stained with the above-mentioned vasculature cells and a sliced specimen stained with cell nuclei, and counting the number of overlapping cell nuclei. The number of cells in the vascular system can be calculated, and the volume can be obtained by calculating the area of the center using ImageJ and multiplying the thickness of the sliced specimen.

  In the present specification, vascular cells mean cells associated with angiogenesis, and are cells constituting blood vessels and blood, and precursor cells and somatic stem cells that can differentiate into the cells. Here, vascular cells are not naturally differentiated into cells that constitute blood vessels and blood such as ES cells, GS cells, iPS cells and other universal cells, and mesenchymal stem cells (MSCs). Things are not included. The vascular cells are preferably cells that constitute blood vessels. Among vertebrate-derived cells (particularly human-derived cells), examples of the cells constituting blood vessels include vascular endothelial cells and vascular smooth muscle cells. Vascular endothelial cells include both venous and arterial endothelial cells. Vascular endothelial progenitor cells can be used as progenitor cells for vascular endothelial cells. Vascular endothelial cells and vascular endothelial progenitor cells are preferred. As cells constituting blood, blood cells can be used, white blood cells such as lymphocytes and neutrophils, monocytes, and hematopoietic stem cells which are stem cells thereof can be used. Moreover, in this specification, a non-vascular cell means cells other than said vascular system. For example, ES cells, iPS cells, mesenchymal stem cells (MSC), myocardial stem cells, cardiomyocytes, fibroblasts, myoblasts, chondrocytes, myoblasts, hepatocytes or nerve cells can be used. Preferably, mesenchymal stem cells (MSC), chondrocytes, myoblasts, myocardial stem cells, cardiomyocytes, hepatocytes or iPS cells can be used. More preferably, they are mesenchymal stem cells (MSC), cardiac stem cells, cardiac muscle cells or myoblasts.

  In the cell transplant cell structure of the present invention, the cell transplant cell structure of the present invention includes two or more types of cells and includes both non-vascular cells and vascular cells. Including those formed. The preferred range of “the cell structure for cell transplantation of the present invention comprising two or more types of cells and containing both non-vascular cells and vascular cells” is the same as described above. Examples of a method for constructing a blood vessel include a method in which a cell sheet in which vascular cells are mixed is attached to a gel material obtained by hollowing out a blood vessel portion in a tunnel shape, and the culture is performed while flowing a culture solution through the tunnel. A blood vessel can also be constructed by sandwiching vascular cells between cell sheets.

(3) Cell structure In the present invention, using the above-described polymer block having bioaffinity and the above-described cell, a plurality of the polymer blocks are mosaic-liked in a space between a plurality of cells. By arranging in a three-dimensional manner, it is possible to have a thickness suitable for cell transplantation, and the polymer blocks and cells having biocompatibility are arranged three-dimensionally in a mosaic manner, thereby providing a structure. A three-dimensional cell structure in which cells uniformly exist in the body is formed, and nutrition can be delivered from the outside to the inside of the three-dimensional cell structure. Thereby, when cell transplantation is performed using the cell structure for cell transplantation of the present invention, necrosis of the transplanted cells is suppressed and transplantation becomes possible. Here, “suppression of necrosis” means that the degree of necrosis is low as compared with the case of transplanting only cells without using the cell structure of the present invention.

  The thickness or diameter of the cell structure for cell transplantation of the present invention can be set to a desired thickness by the method described later in the present specification, but the lower limit is preferably 215 μm or more, and more preferably 400 μm or more. Preferably, it is most preferably 730 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 to 3 cm, more preferably 500 μm to 2 cm, and still more preferably 720 μm to 1 cm. In Example, a cell structure (FIG. 3) having a thickness of 720 μm or more was prepared and then fused to prepare a cell structure having a thickness of 813 μm (FIG. 10). The cell structure for cell transplantation of the present invention is characterized in that a region composed of polymer blocks 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”.

  When the cell structure of the present invention is used as a cell structure before fusion or a cell structure before addition of the second polymer block in the method for producing a cell structure for cell transplantation of the present invention described later The range of the thickness or diameter of the cell structure is preferably 10 μm or more and 1 cm or less, more preferably 10 μm or more and 2000 μm or less, further preferably 15 μm or more and 1500 μm or less, and most preferably 20 μm or more and 1300 μm or less.

  In the cell structure for cell transplantation of the present invention, the ratio of cells to polymer blocks is not particularly limited, but preferably the ratio of polymer blocks per cell is preferably 0.0000001 μg or more and 1 μg or less. Preferably it is 0.000001 microgram or more and 0.1 microgram or less, More preferably, it is 0.00001 microgram or more and 0.01 microgram or less, Most preferably, it is 0.00002 microgram or more and 0.006 microgram or less. From this range, the cells can be present more uniformly. In addition, by setting the lower limit to the above range, the effect of cells can be exerted when used for the above applications, and by setting the upper limit to the above range, any component in the polymer block that is present arbitrarily can be added to the cells. Can supply. Here, the component in the polymer block is not particularly limited, and examples thereof include components contained in the medium described later.

(4) Method for Producing Cell Structure The cell structure of the present invention can be produced by alternately arranging a mass (“block”) made of a polymer material having biocompatibility and cells. The production method is not particularly limited, but is preferably a method of seeding cells after forming a polymer block. Specifically, the cell structure of the present invention can be produced by incubating a mixture of a biocompatible polymer block and a cell-containing culture solution. For example, cells and a polymer block having biocompatibility prepared in advance are arranged in a mosaic pattern in a container and in a liquid held in the container. As a means of arrangement, it is preferable to promote and 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 to be used is preferably a container made of a low cell adhesion material or a cell non-adhesive material, and 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.

The mosaic cell structure obtained by the above method is, for example,
(1) Fusing separately prepared mosaic cell masses, or (2) Volume up under differentiation medium or growth medium,
A cell structure of a desired size can be produced by such a method. The fusion method and the volume increase method are not particularly limited.

  For example, in the step of incubating a mixture of a biocompatible polymer block and a cell-containing culture solution, the cell structure can be increased in volume by replacing the medium with a differentiation medium or a growth medium. Preferably, in the step of incubating the mixture of the biocompatible polymer block and the cell-containing culture solution, a cell structure having a desired size is obtained by further adding the biocompatible polymer block. Thus, a cell structure in which cells are uniformly present in the cell structure can be produced.

  Specifically, the method of fusing separately prepared mosaic cell masses includes a plurality of polymer blocks having a biocompatibility and a plurality of cells, and formed by the plurality of cells. A method for producing a cell structure, comprising a step of fusing a plurality of cell structures in which one or a plurality of the polymer blocks are arranged in part or all of the plurality of gaps.

  “Polymer block having biocompatibility (type, size, etc.)”, “cell”, “gap between cells”, “cell structure obtained (size, etc.) according to the method for producing a cell structure of the present invention The preferred range such as “)” and “ratio of cells to polymer block” is the same as the preferred range for the cell structure of the present invention.

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

  The method for producing a cell structure having a desired size by further adding the above-described polymer block having biocompatibility specifically includes a plurality of first high-affinity cells having biocompatibility. A cell structure comprising a molecular block and a plurality of cells, wherein one or a plurality of the polymer blocks are disposed in part or all of a plurality of gaps formed by the plurality of cells. Furthermore, it is a manufacturing method of a cell structure including the process of adding a 2nd polymer block and incubating. Here, “polymer block having biocompatibility (type, size, etc.)”, “cell”, “gap between cells”, “obtained cell structure (size, etc.)”, “cell and polymer A suitable range such as “block ratio” is the same as the preferred range relating to the cell structure of the present invention.

  Here, the cell structures to be fused are preferably placed at a distance of 0 to 50 μm, more preferably 0 to 20 μm, and still more preferably 0 to 5 μm. When cell structures are fused together, it is conceivable that the cells or the substrate produced by the cells by the growth and expansion of the cells will act as an adhesive and be joined. Becomes easy.

  The size of the first polymer block according to the present invention is preferably 1 μm to 700 μm, more preferably 10 μm to 700 μm, still more preferably 10 μm to 300 μm, and further preferably 20 μm to 150 μm. Or less, particularly preferably 25 μm or more and 106 μm or less. The size of the second polymer block according to the present invention is also preferably 1 μm to 700 μm, more preferably 10 μm to 700 μm, still more preferably 10 μm to 300 μm, and further preferably 20 μm to 150 μm. Or less, particularly preferably 25 μm or more and 106 μm or less.

  The range of the thickness or diameter of the cell structure obtained by the method for producing a cell structure of the present invention 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 pace at which the second polymer block is added when the second polymer block is further added to the cell structure and incubated can be appropriately selected according to the growth rate of the cells to be used. preferable. Specifically, if the pace at which the second polymer block is added is fast, the cells move to the outside of the cell structure, resulting in poor cell uniformity, and if the pace of addition is slow, the percentage of cells increases. Therefore, the cell uniformity is lowered, and therefore, the cell growth rate to be used is taken into consideration.

  As described above, even if the cell structure for cell transplantation of the present invention is a cell structure that does not contain vascular cells, when cell transplantation is performed using the cell structure for cell transplantation of the present invention, Later, at the transplant site, blood vessels can be formed, and when both non-vascular cells and vascular cells are included, compared to a cell structure composed only of non-vascular cells. It becomes possible to form more blood vessels.

As a method for producing a cell structure in the case of containing both non-vascular cells and vascular cells, for example, the following production methods (1) to (3) can be preferably mentioned.
(1) is a production method including a step of adding a vascular cell and a polymer block after forming a cell structure by the above-described method using non-vascular cells. Here, the “vascular cells and polymer block step” includes both the above-described method of fusing the prepared mosaic cell masses together and the method of increasing the volume under differentiation medium or growth medium. . By this method, (i) the area of the non-vascular cell is larger in the central part of the cell structure than the vascular cell, and the peripheral part is compared with the non-vascular cell in the peripheral part. (Ii) a cell structure for cell transplantation in which the area of the non-vascular cell in the central part is larger than the area of the non-vascular cell in the peripheral part, (ii) iii) It is possible to produce a cell structure for cell transplantation in which the area of cells in the central vascular system of the cell structure is smaller than the area of cells in the peripheral vascular system.
(2) is a production method comprising a step of adding a non-vascular cell and a polymer block after forming a cell structure by the above-described method using vascular cells. Here, the “non-vascular cell and polymer block step” includes both the above-described method of fusing the prepared mosaic cell masses and the method of increasing the volume under a differentiation medium or a growth medium. It is a waste. With this method, (i) the area of the vascular system cell is larger in the central part of the cell structure than the non-vascular system cell, and in the peripheral part, the non-vascular system is compared with the vascular system cell. A cell structure having a large cell area, (ii) a cell structure for cell transplantation, wherein the cell area of the central vascular system is larger than the area of the peripheral vascular cell, (iii) It is possible to manufacture a cell structure for cell transplantation in which the area of non-vascular cells in the central part of the cell structure is smaller than the area of non-vascular cells in the peripheral part.
(3) is a production method in which a non-vascular cell and a vascular cell are used substantially simultaneously to form a cell structure by the method described above. In this method, it is possible to produce a cell structure in which any of non-vascular cells and vascular cells are not unevenly distributed at any part of the cell structure.

From the viewpoint of forming blood vessels at the transplantation site after transplantation, the central part of the cell structure has a larger area of vascular cells than non-vascular cells, and the peripheral part is compared with vascular cells. A cell structure having a large area of non-vascular cells or a cell structure for cell transplantation in which the area of cells in the central vascular system is larger than the area of cells in the peripheral vascular system. Preferably, by forming the cell structure, angiogenesis can be further promoted. Furthermore, in the said cell structure, the one where there are many cells which exist in center part can promote angiogenesis more.
For the same reason, a production method comprising a step of adding a non-vascular cell and a polymer block after forming a cell structure with vascular cells is preferred. It is more preferable to increase the number of cells in the vascular system.

  The cell structure for cell transplantation of the present invention can be used for the purpose of cell transplantation at a diseased site such as heart disease such as severe heart failure or severe myocardial infarction or cerebral ischemia / cerebral infarction. It can also be used for diseases such as diabetic kidney, pancreas, peripheral nerves, eyes, and limb circulation disorders. As an implantation method, an incision, an injection, an endoscope, or the like can be used. Unlike the cell transplant such as a cell sheet, the cell structure of the present invention can reduce the size of the structure, and thus enables a minimally invasive transplantation method such as transplantation by injection.

  The cell transplantation method of the present invention includes a biocompatible polymer block that is the cell structure for cell transplantation of the present invention and at least one kind of cell, and a gap between the plurality of cells. And a cell structure for cell transplantation in which a plurality of the polymer blocks are arranged. The preferred range of the cell structure for cell transplantation is the same as described above.

The cell aggregate for cell transplantation of the present invention is a cell aggregate for cell transplantation composed of non-vascular cells and vascular cells,
(1) the area of the vascular system cells in the center of the cell assembly has a region larger than the area of the peripheral vascular cells; and (2) the cell density of the vascular system in the center is 1. Having a region of 0 × 10 ⁻⁴ cells / μm ³ or more,
Is characterized by satisfying at least one of the requirements.
Here, the cell aggregate for cell transplantation according to the present invention refers to a cell transplant containing cells as structural components, and does not exclude inclusion of other components. The cell assembly for cell transplantation of the present invention does not include the polymer block according to the cell structure of the present invention, even if it includes the polymer block according to the cell structure of the present invention. Also good.
Although it does not specifically limit as a shape of a cell aggregate, A sheet form and a spherical lump can be mentioned suitably. Specifically, a cell sheet, a laminate of a plurality of cell sheets, a cell mass (spheroid), and a material in which a plurality of cell masses are fused can be exemplified.
Non-vascular cells and vascular cells are the same as described above.

The central part of the cell aggregate is an area that is 80% of the distance from the center to the surface of the cell aggregate. The peripheral part of the cell aggregate is a place that is 80% from the center. To the surface of the structure. The central part of the cell aggregate is determined as follows.
In an arbitrary cross-section passing through the center of the cell structure, the center of the circle with the radius X is moved along the extension of the cross-section, and the area of the portion excluding the overlapped portion of the moved circle and the cross-section is The radius X is calculated so that it is 64% of the area. The center of the circle with the radius X is moved once, and the portion excluding the overlapped portion of the moved circle and the cross section is taken as the center of the cell assembly. At this time, the cross section having the largest cross-sectional area is most preferable. Note that the center of the cell structure is a portion of the cross section having the maximum cross-sectional area, in which the center of the circle with the radius Y is moved around the extension of the cross section and the overlapped portion of the moved circle and the cross section is excluded. The radius Y determined to be a single point is obtained. The center of the circle with the radius Y is moved once, and one point excluding the overlapped portion between the moved circle and the cross section is defined as the center of the cell structure. When a line segment is formed without being determined as one point, or when there are a plurality of line segments, each line segment is centered on a point that bisects its length.
A cell structure in which the central portion obtained as described above has a distance of 10 μm or more from the outermost contour of the cell aggregate is defined as a target cell aggregate of the present invention.

  Specifically, it is preferable that the ratio of the vascular cells in the central part is 60% to 100% with respect to the total area of vascular cells, more preferably 65% to 100%, 80 % To 100% is more preferable, and 90% to 100% is more preferable. By setting it as the said range, angiogenesis can be promoted more.

In addition, it is preferable that the cell aggregate has a region where the cell density of the vascular system in the central part is 1.0 × 10 ⁻⁴ cells / μm ³ or more, and the cell density is the entire cell aggregate in the central part. It is more preferable. Here, having a region that is 1.0 × 10 ⁻⁴ cells / μm ³ or more specifically refers to a sample having a region of the density when an arbitrary 2 μm sliced sample is prepared. Say that exists. The cell density is more preferably 1.0 × 10 ^{−4 to} 1.0 × 10 ⁻³ cells / μm ³ , further preferably 1.0 × 10 ^{−4 to} 2.0 × 10 ⁻⁴ cells / μm ^3. More preferably, it is 1.1 × 10 ^{−4 to} 1.8 × 10 ⁻⁴ cells / μm ³ , and further preferably 1.4 × 10 ^{−4 to} 1.8 × 10 ⁻⁴ cells / μm ³ . By setting it as the said range, angiogenesis can be promoted more. In addition, it is preferable to satisfy both requirements (1) and (2).

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

Example 1 Recombinant Peptide CBE3 described below was prepared as a recombinant peptide (recombinant peptide) (described in WO2008-103041).
CBE3
Molecular weight: 51.6kD
Structure: GAP [(GXY) 63] 3G
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 in WO2008 / 103041, except that X at the end is corrected to “P”)
GAP (GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGAPG

Example 2 Production of Recombinant Peptide μ Block An amorphous μ block was produced using the recombinant peptide CBE3 as a base block. Dissolve 1000 mg of recombinant peptide in 9448 μL of ultrapure water, add 152 μL of 1N HCl, add 400 μL of 25% glutaraldehyde to a final concentration of 1.0%, react at 50 ° C. for 3 hours, and crosslink A gelatin gel was prepared. This crosslinked gelatin gel was immersed in 1 L of 0.2 M glycine solution and shaken at 40 ° C. for 2 hours. Thereafter, the crosslinked gelatin gel was washed by shaking in 5 L of ultrapure water for 1 hour, the ultrapure water was replaced with a new one, and the washing was repeated again for 1 hour, for a total of 6 washes. The cross-linked gelatin gel after washing was frozen at −80 ° C. for 5 hours, and then freeze-dried with a freeze-dryer (EYELA, FDU-1000). The obtained freeze-dried product was pulverized with New Power Mill (Osaka Chemical, 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 recombinant peptide μ blocks of 25 to 53 μm and 53 to 106 μm.

Example 3 Preparation of Natural Gelatin μ Block An amorphous μ block was prepared using natural gelatin (Nippi, Nippi Gelatin High Grade Gelatin APAT) as a base block. Dissolve 1000 mg of natural gelatin in 9448 μL of ultrapure water, add 152 μL of 1N HCl, add 400 μL of 25% glutaraldehyde to a final concentration of 1.0%, react at 50 ° C. for 3 hours, and crosslink A gelatin gel was prepared. This crosslinked gelatin gel was immersed in 1 L of 0.2 M glycine solution and shaken at 40 ° C. for 2 hours. Thereafter, the crosslinked gelatin gel was washed by shaking in 5 L of ultrapure water for 1 hour, the ultrapure water was replaced with a new one, and the washing was repeated again for 1 hour, for a total of 6 washes. The cross-linked gelatin gel after washing was frozen at −80 ° C. for 5 hours, and then freeze-dried with a freeze-dryer (EYELA, FDU-1000). The obtained freeze-dried product was pulverized with New Power Mill (Osaka Chemical, 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 natural gelatin μ blocks of 25 to 53 μm and 53 to 106 μm.

Example 4 Production of Mosaic Cell Mass Using Recombinant Peptide μ Block Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 500,000 cells / mL with a growth medium (Takara Bio: MSCGM-CD ^™ BulletKit ^™ ). After adding the recombinant peptide μ block prepared in Example 2 to 1.0 mg / mL, 100 μL was seeded on a Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom U-shaped) and allowed to stand for 18 hours. Thus, a spherical mosaic cell mass composed of a recombinant peptide μ block having a diameter of about 1 mm and hMSC cells was prepared (0.002 μg polymer block per cell). Thereafter, the medium was replaced with cartilage differentiation medium (Takara Bio: hMSC Differentiation BulletKit ^™ , Chondrogenic, TGF-β3) (200 μL). On Day 7, a mosaic cell mass was formed in a spherical shape having a diameter (= thickness) of 1.54 mm (FIG. 1). In addition, since this mosaic cell mass is produced in a U-shaped plate, it is produced in a spherical shape. Medium exchange was performed at Day 3, 7, 10, 14, 17, 21.

Example 5: Production of Mosaic Cell Mass Using Natural Gelatin µ Block Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 500,000 cells / mL with a growth medium (Takara Bio: MSCGM-CD ^™ BulletKit ^™ ). After adding the natural gelatin μ block prepared in Example 3 to 1.0 mg / mL, 100 μL was seeded on a Sumilon Celtite X96U plate, left to stand for 18 hours, and spherical, natural, about 1 mm in diameter. A mosaic cell mass composed of gelatin μ block and hMSC cells was prepared (0.002 μg polymer block per cell). Thereafter, the medium was replaced with cartilage differentiation medium (Takara Bio: hMSC Differentiation BulletKit ^™ , Chondrogenic, TGF-β3) (200 μL). On Day 7, the mosaic cell mass was formed into a spherical shape with a diameter (= thickness) of 1.34 mm (FIG. 2). In addition, since this mosaic cell mass is produced in a U-shaped plate, it is produced in a spherical shape.

Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 500,000 cells / mL with a growth medium (Takara Bio: MSCGM-CD ^™ BulletKit ^™ ), and the natural gelatin μ block prepared in Example 3 was adjusted to 0 at the final concentration. .005 mg / mL, 0.01 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 1.0 mg / mL, 2.0 mg / mL), and then 100 μL of Sumilon It seed | inoculated to the Celtite X96U plate, and left still for 18 hours, The diameter 1mm diameter and spherical mosaic cell mass was able to be produced (0.00001, 0.0002, 0.0004, 0.002, 0.00. 004 μg polymer block).

Example 6: Sample analysis A tissue section was prepared for the mosaic cell mass using the recombinant peptide μ block prepared in Example 4. To the mosaic cell mass in the medium produced in Example 4, after removing the medium, 200 μL of PBS was added and washed to remove PBS. After this washing step was repeated twice, the washed mosaic cell mass was immersed in 10% formalin and formalin fixation was performed for 2 days. Thereafter, it was embedded in paraffin to prepare a tissue section. The sections were stained with HE (hematoxylin and eosin staining), and the state of the cells and the gelatin μ block was analyzed. The results are shown in FIG. 3, FIG. 4 and FIG. As a result, it was confirmed that a gelatin μ block and a three-dimensional structure in which cells were arranged in a mosaic shape were produced, and that the cells were present in a mosaic cell mass in a normal state. Further, it was shown that a mosaic cell mass having a thickness of at least 720 μm or more could be produced from this cross section.

Example 7: Fusion of mosaic cell mass Whether the mosaic cell mass produced in Example 4 can be fused, that is, by arranging mosaic cell masses, they can be naturally fused to form a larger tertiary structure. It carried out about. Two, three, and four mosaic cell masses from Day 6 prepared in Example 4 were arranged in a Sumilon Celtite X96U plate and cultured for 5 days. As a result, it was clarified that the mosaic cell mass naturally fuses when the cells arranged on the outer periphery are joined between the mosaic cell masses. FIG. 6 shows a photograph taken with a stereomicroscope. In the mosaic cell mass on the fusion start date (denoted as Day 6), the mosaic cell clusters are merely arranged next to each other, but on the fifth day (denoted as Dya11) from the start of the fusion, You can see how new layers are formed and fused. 7, 8, 9, 10, and 11 show the results of preparing a tissue section and HE staining for the cross section of the fused mosaic cell mass (fixed is 10% formalin, embedded is paraffin embedded). . It can be seen that a fusion layer is formed between the mosaic cell masses of the cells and the extracellular matrix produced by the cells, and the mosaic cell masses are fused and bound together. Thereby, it was shown that the mosaic cell mass produced in the present invention can be naturally fused, and a larger structure can be formed by fusing. Therefore, it can be seen that by using the present invention, it is possible to produce a cell sheet having a thickness or to produce a more three-dimensional three-dimensional structure.

Example 8 Production of Mosaic Cell Mass under Growth Medium Using Recombinant Peptide μ Block Human bone marrow-derived mesenchymal stem cells (hMSC) were grown in growth medium (Takara Bio: MSCGM-CD ^™ BulletKit ^™ ) at 500,000 cells / After adjusting to mL and adding the recombinant peptide μ block prepared in Example 2 to 1.0 mg / mL, 100 μL was seeded on a Sumilon Celtite X96U plate, allowed to stand for 18 hours, and spherical with a diameter of 1 mm. Mosaic cell masses were prepared (0.002 μg polymer block per cell). Thereafter, the medium was increased to 200 μL, and the medium was changed every three days for cultivation. On Day 7, a mosaic cell mass was formed in a spherical shape having a diameter (= thickness) of 1.34 mm (this mosaic cell mass was produced in a spherical shape because it was produced in a U-shaped plate). Sectional photographs of the Day 7 mosaic cell mass are shown in FIGS. It can be seen that on the cross-section, a thickness of at least 624 μm or more is achieved even at a thin portion.

Example 9: Volume up of mosaic cell mass (under growth medium)
For the mosaic cell mass on Day 3 (Day 3) prepared in Example 8, 0.1 mg of the recombinant peptide μ block prepared in Example 2 was added to the growth medium (Takara Bio: MSCGM-CD ^™ BulletKit) when the medium was replaced. ^TM ) was added in suspension. Thereafter, 0.1 mg of recombinant peptide μ block was added in accordance with the medium exchange of Day 7, 10, 14, 17, 21.

The diameter of the mosaic cell mass observed with a stereomicroscope (calculated as the average of two different diameters), the area of the captured mosaic cell mass, and the calculated volume (calculated from the diameter obtained above by 4 / 3πr ³ ), The change with time for each is shown in FIGS. As a result, it was finally formed into a spherical shape with an average diameter (= thickness) of 3.41 mm on Day 21. Thereby, it can be seen that a mosaic cell mass having a size of at least up to 3.41 mm can be produced. It can also be seen that the size can be increased by continuing the volume increase by this method.

  Tissue sections (HE staining) at this time are shown in FIGS. It can be seen that cells and recombinant peptide μ blocks are arranged in a mosaic pattern. In addition, since the mosaic cell mass is 3 mm in size and small as a specimen, it was extremely difficult to make a cross section at the center of the sphere. Therefore, even though the deepest part of the sphere is not obtained as a section, it can be seen that the thickness of the part of the specimen from which the section is collected is at least 1.17 mm.

  When the culture medium was replaced on Day 7, 10, 14, 17, and 21, the diameter was not changed as shown in FIGS. 12, 13, and 14 when cultured without adding the recombinant peptide μ block. When cultured without adding the recombinant peptide μ block, a layered structure of only cells and the extracellular matrix produced by the cells is formed by the proliferated cells in the outermost layer of the mosaic cell mass. As a result, a state in which the diffusion of nutrients is blocked by the layer of the cells and the production extracellular matrix is formed, and the cells can no longer proliferate (the sphere becomes larger). On the other hand, when a recombinant peptide μ block is added at any time according to the medium exchange, the recombinant peptide μ block is always fitted in a mosaic with the proliferated cells, so that even if the cells proliferate, It becomes possible to maintain the mosaic structure composed of the recombinant peptide μ block. As a result, the supply route of nutrients provided by the recombinant peptide μ block is always secured, the outer shell layer formed by the cells and the produced extracellular matrix is not generated, and the mosaic cell mass can be enlarged.

Example 10: Volume increase of mosaic cell mass (under cartilage differentiation medium)
For the mosaic cell mass on Day 3 (Day 3) prepared in Example 4, the 0.1 mg recombinant peptide μ block (0.1 mg) prepared in Example 2 was replaced with the cartilage differentiation medium (Takara Bio: hMSC Differentiation BulletKit ^™ , Chondrogenic, TGF-β3) was added in suspension. Thereafter, 0.1 mg of recombinant peptide μ block was added in accordance with the medium exchange of Day 7, 10, 14, 17, 21.

The diameter of the mosaic cell mass observed with a stereomicroscope (calculated as the average of two different diameters), the area of the captured mosaic cell mass, and the calculated volume (calculated from the diameter obtained above by 4 / 3πr ³ ), The change with time for each is shown in FIGS. As a result, it was finally formed into a spherical shape with an average diameter (= thickness) of 2.05 mm on Day 21. Thereby, it can be seen that a mosaic cell mass having a size of at least 2.05 mm can be produced. It can also be seen that the size can be increased by continuing the volume increase by this method.

  Tissue sections (HE staining) at this time are shown in FIGS. It can be seen that cells and recombinant peptide μ blocks are arranged in a mosaic pattern. Further, since the mosaic cell mass is 2 mm in size and small as a specimen, it was extremely difficult to make a cross section at the center of the sphere. Therefore, even though the deepest part of the sphere is not obtained as a section, it can be seen that the part of the specimen from which the section is collected has a thickness of at least 897 μm.

  When the culture medium was replaced on Day 7, 10, 14, 17, and 21, the diameter was not changed as shown in FIGS. 12, 13, and 14 when cultured without adding the recombinant peptide μ block. When cultured without adding the recombinant peptide μ block, a layered structure of only cells and the extracellular matrix produced by the cells is formed by the proliferated cells in the outermost layer of the mosaic cell mass. This is because the layer of the cell and the production extracellular matrix forms a state in which the diffusion of nutrients is prevented, and the cell cannot further grow (the sphere becomes larger). On the other hand, when a recombinant peptide μ block is added at any time according to the medium exchange, the recombinant peptide μ block is always fitted in a mosaic with the proliferated cells, so that even if the cells proliferate, It is possible to continue to maintain a mosaic structure composed of gelatin μ blocks. As a result, the supply route of nutrients provided by the recombinant peptide μ block is always secured, the outer shell layer formed by the cells and the produced extracellular matrix is not generated, and the mosaic cell mass can be enlarged.

Example 11: Quantification of GAG production amount of mosaic cell mass (change over time)
Mosaic cell mass produced in Example 4 and Example 5 (hMSC cell + recombinant peptide, hMSC cell + natural gelatin), and cell mass produced only with cells (in the same manner as in Example 4, without gelatin block) The amount of glycosaminoglycan in the mosaic cell mass was quantified. The measurement is carried out by the method using Dimethylmethylene blue dye (Farndale et al., Improved quantitation and sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochimica et Biophysica Acta 883 (1986) 173-177). Biochemical Biobusiness). Absorbance at 530 nm was measured and quantified. As shown in FIG. 21, it has been confirmed that a characteristic absorption peak is observed at 525-530 nm by this method.

  The results of quantifying the amount of GAG over time are shown in the graph of FIG. As a result, in the cell mass produced without the gelatin μ block or recombinant peptide μ block, the amount of glycosaminoglycan (GAG) produced is low, whereas the mosaic cell mass produced with the natural gelatin μ block, For the mosaic cell mass produced with the recombinant peptide μ block, the amount of GAG produced was extremely high. Thereby, in the mosaic cell mass produced in Example 4 and Example 5, the cartilage differentiation was promoted, and the produced mosaic cell mass had a function as a cell (having GAG production ability). I was able to confirm. Furthermore, the amount of GAG produced was significantly higher in the mosaic cell mass prepared by inserting the recombinant peptide μ block than in the mosaic cell mass prepared by adding the natural gelatin μ block. As a result, it was found that the mosaic cell mass produced by inserting the recombinant peptide μ block can maintain higher cell activity and substrate production activity than the natural gelatin μ block. It was shown that this can be achieved by using a recombinant peptide.

Example 12: ATP quantification of mosaic cell mass The amount of ATP (adenosine triphosphate) produced and retained by cells in each mosaic cell mass was quantified. ATP is known as an energy source for all living organisms. By quantifying the amount of ATP synthesized and retained, the state of metabolic activity and activity of cells can be known. CellTiter-Glo (Promega) was used for the measurement. For comparison, the mosaic cell mass produced in Example 4 and Example 5 (hMSC cell + recombinant peptide, hMSC cell + natural gelatin), and the cell mass produced only with cells (in the same manner as in Example 4, gelatin block) The ATP content in each mosaic cell mass was quantified using CellTiter-Glo.

  The results are shown in FIG. Thus, it was found that the mosaic cell mass produced using the recombinant peptide μ block or the gelatin μ block had significantly higher ATP production / retention amount than the cell mass produced only with the cells (p <0.01). ). This is because when the μ block is fitted in a mosaic shape, a nutrient supply route into the mosaic cell mass by the μ block is provided, and the state in which the metabolic activity of the cell is higher than that of the cell-only mass is maintained. Suggests. Further, it was found that the amount of ATP produced / retained was significantly higher in the mosaic cell mass prepared by inserting the recombinant peptide μ block than in the mosaic cell mass prepared by adding the natural gelatin μ block. As a result, it was found that the mosaic cell mass prepared by inserting the recombinant peptide μ block had higher cell survival than the natural gelatin μ block, and the cells were alive inside. It has been shown that improved cell survival, which was impossible with natural gelatin, can be achieved by using recombinant peptides.

Example 13: Preparation of PLGA µ block PLGA (lactic acid / glycolic acid copolymer: Wako, PLGA7520) 0.3 g was dissolved in dichloromethane (3 mL). The approximately PLGA solution was vacuum dried with a dryer (EYELA, FDU-1000) to obtain a dried product of PLGA. The dried PLGA was pulverized with New Power Mill (Osaka Chemical, New Power Mill PM-2005). The pulverization was performed by pulverization at a maximum rotation speed of 10 seconds × 20 times. The obtained pulverized product was sized with a stainless steel sieve to obtain PLGA μ blocks of 25 to 53 μm and 53 to 106 μm.
PLGA: “1 / IOB” Value: 0.0552

Example 14 Preparation of Mosaic Cell Mass Using PLGA Example of adjusting human bone marrow-derived mesenchymal stem cells (hMSC) to 500,000 cells / mL with growth medium (Takara Bio: MSCGM-CD ^™ BulletKit ^™ ) After adding the PLGAμ block prepared in Step 13 (prepared by changing the conditions so that the final concentrations were 0.1 mg / mL, 0.2 mg / mL, 1.0 mg / mL, and 2.0 mg / mL), 100 μL Was seeded on a Sumilon Celtite X96U plate and allowed to stand for 18 hours to produce a spherical mosaic cell mass with a diameter of less than 1 mm and a spherical shape (0.0002, 0.0004, 0.002, 0.004 μg per cell). Molecular block). Thereafter, the medium was increased to 200 μL, and the medium was changed every three days for cultivation. In addition, since this mosaic cell mass is produced in a U-shaped plate, it is produced in a spherical shape. A stereomicrograph of the Day 2 PLGA mosaic cell cluster is shown in FIG.

Example 15: Preparation of agarose μ block 5 g of agarose powder was added to ultrapure water (100 mL), and heated and dissolved using a microwave oven. The obtained 5% agarose solution is returned to room temperature to form a solid, frozen at −80 ° C. for 5 hours, and then freeze-dried with a freeze dryer (EYELA, FDU-1000). A lyophilized product was obtained. The agarose freeze-dried product was pulverized with New Power Mill (Osaka Chemical, New Power Mill PM-2005). The pulverization was performed by pulverization at a maximum rotation speed of 10 seconds × 20 times. The obtained pulverized product was sized with a stainless steel sieve to obtain 25-53 μm and 53-106 μm agarose μ blocks.
IOB value: 3.18

Example 16 Production of Mosaic Cell Mass Using Agarose A human bone marrow-derived mesenchymal stem cell (hMSC) was adjusted to 500,000 cells / mL with a growth medium (Takara Bio: MSCGM-CD ^™ BulletKit ^™ ). 15 was added (prepared by shaking the conditions so that the final concentration was 0.1 mg / mL and 1.0 mg / mL), 100 μL was seeded on a Sumilon Celtite X96U plate, and 18 The mixture was allowed to stand for a period of time, and a spherical mosaic cell mass having a diameter of less than 1 mm and a spherical shape was produced (0.0002, 0.002 μg polymer block per cell). Thereafter, the medium was increased to 200 μL, and the medium was changed every three days for cultivation. In addition, since this mosaic cell mass was produced in a U-shaped plate, it was produced in a spherical shape.

Example 17: Preparation of Mosaic Cell Mass Using Cardiomyocytes Neonatal SD rat cardiomyocytes (rCMC) were made to 500,000 cells / mL in cardiomyocyte culture medium (Primary Cell Co., Ltd: CMCM cardiomyocyte culture medium). After adjusting and adding the recombinant peptide μ block prepared in Example 2 to 0.5, 1.0, and 3.0 mg / mL, 100 μL of each was added to Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom with U And then left to stand for 18 hours to prepare a mosaic cell mass composed of a recombinant peptide μ block having a diameter of about 1 to 2 mm and rCMC cells (0.001, 0.002, 0.006 μg per cell). Polymer block). Medium exchange was performed at Day 3, 7, 10, 14, 17, 21.

  It was confirmed at the stage of Day 1 and Day 3 that the rCMC mosaic cell mass was already synchronously pulsating as the whole structure (FIG. 25). In the specification, since it is difficult to show a moving image, FIG. 25 shows an image obtained by capturing the same spot 0.2 seconds after the moving image as a still image. If you look at the part marked with a triangle, you can see that the whole is moving with two pictures.

  As a result, it was confirmed that the cell three-dimensional structure (mosaic cell mass) of the present invention can be formed even using cardiomyocytes, and in the mosaic cell mass using cardiomyocytes, the entire structure is synchronized. It was proved that a cell structure that can be beaten was obtained.

Example 18 Production of Mosaic Cell Mass Using GFP-Expressing HUVEC (Human Umbilical Vein Endothelial Cells) Human umbilical vein endothelial cells expressing GFP (GFP-HUVEC: Angio-Proteomie) were used as an endothelial cell medium (Kurabo). : Medium 200S, LSGS, antibacterial agent GA solution) was adjusted to 500,000 cells / mL, and the recombinant peptide μ block prepared in Example 2 was added to 0.3, 1.0, and 3.0 mg / mL. Thereafter, 100 μL of each was seeded on a Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom U-shaped) (polymer block of 0.0006, 0.002, 0.006 μg per cell). Similarly, the cells were adjusted to 1.5 million cells / mL, and the recombinant peptide μ block prepared in Example 2 was added to 1.0 mg / mL, and then 100 μL and 200 μL were added to the Sumilon Celtite X96U plate. What was seeded on (Sumitomo Bakelite, U-shaped bottom) was also produced. About all, it left still for 18 hours and produced the mosaic cell mass which consists of recombinant peptide microblock about 1-2 mm in diameter and a GFP-HUVEC cell. Medium exchange was performed at Day 3, 7, 10, 14, 17, 21.

  FIG. 26 shows micrographs and fluorescence micrographs of 50,000 cells + 0.03 mg mosaic cell mass, 300,000 cells + 0.2 mg mosaic cell mass. Since GFP-HUVEC cells emit GFP fluorescence, it is easy to understand the distribution in the mosaic cell mass using a fluorescence microscope. As a result, it was proved that the three-dimensional cell structure (mosaic cell mass) of the present invention can be prepared using vascular endothelial cells.

  In addition, it was demonstrated that the cell structure (mosaic cell mass) of the present invention can be formed from various cells such as mesenchymal stem cells, cardiomyocytes, and vascular endothelial cells. At the same time, it was also shown that it can be formed using various polymer blocks such as recombinant peptide blocks, animal gelatin blocks, PLGA blocks, and agarose blocks. Thereby, it was proved that the three-dimensional cell structure (mosaic cell mass) of the present invention can be formed in various cell types and various polymer block types.

Example 19- (1): Production of mosaic cell mass using recombinant peptide μ block (hMSC)
Human bone marrow-derived mesenchymal stem cells (hMSC) are adjusted to 100,000 cells / mL in a growth medium (Takara Bio: MSCGM BulletKit ^™ ), and the recombinant peptide μ block prepared in Example 2 is 0.1 mg / mL. After that, 200 μL is seeded on a Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom is U-shaped), centrifuged (600 g, 5 minutes) with a desktop plate centrifuge, allowed to stand for 24 hours, and about 1 mm in diameter. A spherical mosaic cell mass composed of recombinant peptide μ block and hMSC cells was prepared (0.001 μg polymer block per cell). In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical.

Example 19- (2): Enlargement by fusion of mosaic cell masses The mosaic cell mass produced in Example 19- (1) can be fused, that is, naturally fused by arranging the mosaic cell masses. It was carried out whether a larger tertiary structure could be formed. First, a square silicon sheet (3 mm thick) having a size that fits perfectly into a PrimeSurface 90 mm dish was prepared, and the center was cut into a 1.5 cm square. The silicon sheet was disinfected with ethanol and washed with PBS. It was placed in a PrimeSurface 90 mm dish, and 1500 mosaic cell clusters prepared in Example 19- (1) were placed in a 1.5 cm square. 50 ml of growth medium (Takara Bio: MSCGM BulletKit ^™ ) was gently put and cultured for 2 days. As a result, a fusion of 1.5 cm square and 2 mm thick mosaic cell mass could be created. FIG. 27 shows a photograph taken with a stereomicroscope. It was also confirmed from the HE-stained section of the cross section of this fusion that the internal cells were alive. Thus, it was shown that the mosaic cell mass can be naturally fused, and a large structure of cm order can be formed by fusing. Therefore, it can be seen that the mosaic cell mass can be produced in the form of a cell sheet having a thickness that can be used for cell transplantation, or a more three-dimensional three-dimensional structure can be produced.

Example 20: Production of mosaic cell mass using recombinant peptide μ block (hMSC + hECFC)
Example 20- (1): Recombinant peptide μ block prepared in Example 2 by adjusting human bone marrow-derived mesenchymal stem cells (hMSC) to 100,000 cells / mL in a growth medium (Takara Bio: MSCGM BulletKit ^™ ) Was added to a concentration of 0.1 mg / mL, 200 μL was seeded on a Sumilon Celtite X96U plate, centrifuged (600 g, 5 minutes) with a desktop plate centrifuge, allowed to stand for 24 hours, and spherical with a diameter of about 1 mm. A mosaic cell mass consisting of recombinant peptide μ block and hMSC cells was prepared. Thereafter, the medium was removed, human vascular endothelial progenitor cells (hECFC) were adjusted to 100,000 cells / mL with a growth medium (Lonza: EGM-2 + ECFC serum supplement), and the recombinant peptide μ block prepared in Example 2 was reduced to 0. After adding to a dose of 025 mg / mL, 200 μL of hMSC cell mosaic cell mass is seeded on a Sumilon Celtite X96U plate, centrifuged (600 g, 5 minutes) on a desktop plate centrifuge, and allowed to stand for 24 hours. Then, a spherical mosaic cell mass having a diameter of about 1 mm and having a hECFC and recombinant peptide μ block layer formed on the periphery of the mosaic cell mass composed of the recombinant peptide μ block and hMSC cells was prepared. In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical.

Example 20- (2): Human vascular endothelial progenitor cells (hECFC) were adjusted to 100,000 cells / mL with a growth medium (Lonza: EGM-2 + ECFC serum supplement), and the recombinant peptide μ block prepared in Example 2 was used. After adding to 0.05 mg / mL, 200 μL is seeded on a Sumilon Celtite X96U plate, centrifuged with a desktop plate centrifuge (600 g, 5 minutes), allowed to stand for 24 hours, and flat ECFC and recombinant A mosaic cell mass composed of peptide μ blocks was prepared. Thereafter, the medium was removed, and human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 100,000 cells / mL with a growth medium (Takara Bio: MSCGM BulletKit ^™ ), and the recombinant peptide μ block prepared in Example 2 was used. After adding to 0.1 mg / mL, 200 μL of hECFC mosaic cell mass is seeded on a Sumilon Celtite X96U plate, centrifuged (600 g, 5 minutes) with a desktop plate centrifuge, and allowed to stand for 24 hours. A mosaic cell mass composed of a recombinant peptide μ block and hMSC cells including a spherical cell cluster composed of ECFC and a recombinant peptide μ block having a spherical shape of about 1 mm in diameter was prepared. In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical (the mosaic cell mass obtained here is set to A). Furthermore, human vascular endothelial progenitor cells (hECFC) at 200,000 cells / mL, recombinant peptide μ block at 0.1 mg / mL, and human bone marrow-derived mesenchymal stem cells (hMSC) at 200,000 cells / mL, recombinant peptide μ block Was 0.2 mg / mL, a mosaic cell mass having a thickness of about 1 mm and a diameter of about 1.5 mm could be produced (the mosaic cell mass obtained here is designated as B).

Example 20- (3): Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 100,000 cells / mL in a growth medium (Takara Bio: MSCGM BulletKit ^™ ) to proliferate human vascular endothelial progenitor cells (hECFC) After adjusting to 100,000 cells / mL with a medium (Lonza: EGM-2 + ECFC serum supplement) and adding the recombinant peptide μ block prepared in Example 2 to 0.15 mg / mL, 200 μL was added to Sumilon Celtite. It seed | inoculated to X96U plate, centrifuged (600g, 5 minutes) with the tabletop plate centrifuge, and left still for 48 hours, and the mosaic cell mass which consists of spherical recombinant peptide microblock about 1 mm in diameter, hMSC, and hECFC was produced. In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical.

Comparative Example 1: Production of cell mass only of cells (hMSC)
Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 375,000 cells / mL with growth medium (Takara Bio: MSCGM BulletKit ^™ ), and 200 μL was added to Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom U-shaped) ), Centrifuged with a tabletop centrifuge (600 g, 5 minutes), and allowed to stand for 24 hours to produce a spherical cell mass composed of hMSC cells having a diameter of about 1 mm. In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical.

Comparative Example 2: Production of cell mass only of cells (hMSC + hECFC)
Human vascular endothelial progenitor cells (hECFC) were adjusted to 100,000 cells / mL with growth medium (Lonza: EGM-2 + ECFC serum supplement), 200 μL was seeded on Sumilon Celtite X96U plate, and centrifuged with a tabletop centrifuge ( 600 g, 5 minutes) and allowed to stand for 24 hours to prepare a cell cluster of hECFC. Thereafter, the medium was removed, and human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 300,000 cells / mL in a growth medium (Takara Bio: MSCGM BulletKit ^™ ), and 200 μL containing hECFC mosaic cell mass was added to Sumilon cellite. X96U plate was seeded, centrifuged with a tabletop centrifuge (600 g, 5 minutes), and allowed to stand for 24 hours to produce a spherical cell mass composed of spherical hECFC and hMSC having a diameter of about 1 mm (obtained here) A cell mass is designated as A). In addition, human vascular endothelial progenitor cells (hECFC) were adjusted to 200,000 cells / mL with a growth medium (Lonza: EGM-2 + ECFC serum supplement), 200 μL was seeded on a Sumilon celltite X96U plate, and then a desktop plate centrifuge. The mixture was centrifuged (600 g, 5 minutes) and allowed to stand for 24 hours to prepare a cell cluster of hECFC. Thereafter, the medium was removed, and human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 200,000 cells / mL with a growth medium (Takara Bio: MSCGM BulletKit ^™ ), and 200 μL containing hECFC mosaic cell mass was added to Sumilon cellite. X96U plate was seeded, centrifuged with a tabletop centrifuge (600 g, 5 minutes), and allowed to stand for 24 hours to produce a spherical cell mass composed of spherical hECFC and hMSC having a diameter of about 1 mm (obtained here) The cell mass is designated as B).

Specimen analysis Tissue sections were prepared for mosaic cell masses using the recombinant peptide μ blocks prepared in Examples 19- (1) and 20 and Comparative Example 1. The section thickness was 2 μm. To the mosaic cell mass in the prepared medium, the medium was removed, 200 μL of PBS was added and washed, and PBS was removed. After this washing step was repeated twice, the washed mosaic cell mass was immersed in 10% formalin to perform formalin fixation. Thereafter, it was embedded in paraffin to prepare a tissue section. In Example 19- (1) and Comparative Example 1, the sections were stained with HE (hematoxylin and eosin staining), and the state of the cells and the recombinant peptide μ block was analyzed. The results are shown in FIGS. Thereby, in the mosaic cell mass, the recombinant peptide μ block and the three-dimensional structure in which the cells are arranged in a mosaic shape are produced, and further, the cells are present in the mosaic cell mass in a normal state. It could be confirmed. Further, it was shown that a mosaic cell mass and a cell mass having a thickness of at least 500 μm could be produced from the cross section.

Further, in the mosaic cell mass of Example 20, the sections were divided into CD31 antibody (EPT Anti CD31 / PECAM-1) for hECFC staining, and CD29 antibody (EPT Anti Integrin β-1 (CD29)) for hMSC and hECFC staining, respectively. Immunostaining was performed with a kit using DAB color development (Daco LSAB2 kit universal K0673 Daco LSAB2 kit / HRP (DAB) for both rabbit and mouse primary antibodies) (FIGS. 30, 31, 32 and 33). For the mosaic cell mass produced in Example 20- (1) to (3), the ratio of the area of hECFC (vascular cells) in the center using the image processing software ImageJ and the staining method with CD31 antibody described above. Asked. The “center” here is as defined above.
As a result, the ratio of the area of hECFC (vascular cells) in the central part of the mosaic cell mass of Example 20- (1) was 24%, and the central part of the mosaic cell mass of Example 20- (2). The area ratio of hECFC was 91% for both A and B, and the area ratio of hECFC at the center of the mosaic cell mass of Example 20- (3) was 67%.

Further, for the mosaic cell mass of Example 20, the cell density of hECFC present in the center was calculated by superimposing the above-described staining with CD31 antibody and HE staining (hematoxylin and eosin staining). The cell density of the central vascular system can be obtained by actually counting the number of cells in a sliced sample and dividing the number of cells by the volume. First, using Photoshop, the above two images were overlaid, and the number of HE-stained cell nuclei superimposed with the CD31 antibody staining was counted to calculate the number of cells. On the other hand, the volume was determined by calculating the area of the center using ImageJ and multiplying the thickness of the sliced specimen by 2 μm.
As a result, the cell number of hECFC (vascular cells) in the central part of the mosaic cell mass of Example 20- (1) was 1.58 × 10 ⁻⁵ cells / μm ³ , and A of Example 20- (2). The number of hECFC cells at the center of the mosaic cell mass is 1.12 × 10 ⁻⁴ cells / μm ³ , and the number of hECFC cells at the center of the mosaic cell mass of Example 20- (3) is 1.06 × 10 ⁻⁴ cells. / μm ³ . When the number of cells and the block weight were doubled in B of Example 20- (2), the number of hECFC cells in the center was 1.72 × 10 ⁻⁴ cells / μm ³ .

Example 21: Survival difference evaluation test in mouse body using mosaic cell mass of hMSC A test was conducted to confirm that hMSC at the center of the mosaic cell mass was alive in the mouse body.

Mosaic cell mass and transplantation of cell mass Mice were males of Balb / c Nude (Charles River), 5 weeks old, raised for about 5 weeks, and about 10 weeks old were used. First, under anesthesia, the skin between the first and second ankle joints (hereinafter referred to as the lower leg) was incised with scissors from the tip of the mouse foot, and the skin was turned. Thereafter, the lower leg muscle was incised with a scalpel about 5 mm, and the hMSC mosaic cell mass produced in Example 19- (1) and the hMSC cell mass produced in Comparative Example 1 were buried in the incised part using tweezers. The muscle incision was sutured with sutures, and the skin was further sutured.
Furthermore, as another technique, a transplantation method in which muscles were injected into the lower leg was also performed. Ten hMSC mosaic cell masses prepared in Example 19- (1) or 10 hMSC cell masses prepared in Comparative Example 1 were placed in a 1 mm syringe together with 200 μl of hMSC growth medium (Takara Bio: MSCGM BulletKit ^™ ). Injection was made into the muscles of the lower leg with an injection needle (Terumo).

Collection of Mosaic Cell Mass Dissection was performed on the 2nd, 5th, 8th, and 13th days after transplantation. In the case of muscle incision transplantation, the skin of the lower leg of the mouse was peeled off, the suture of the muscle of the lower leg was removed, and the incision was cut open with a scalpel. After visually confirming the transplanted hMSC mosaic cell mass and hMSC cell mass, the thigh was cut with scissors and the tip from the ankle.
In the case of intramuscular injection transplantation, the skin of the lower leg of the mouse was peeled off, and the lower leg muscle was incised with a scalpel. After visually confirming the transplanted hMSC mosaic cell mass and hMSC cell mass, the muscles to which they were attached were cut out.

Sample analysis Tissue sections were prepared for the mosaic cell mass or the lower leg including the cell mass, the hMSC mosaic cell mass and the muscle to which the hMSC cell mass adhered, and the mosaic cell mass and cell mass before transplantation. The thigh was immersed in 4% paraformaldehyde, and formalin fixation was performed. Thereafter, it was embedded in paraffin, and tissue sections of the lower leg including hMSC mosaic cell mass and hMSC cell mass were prepared. The sections were immunostained with CD29 antibody and DAB coloring method for HE staining (hematoxylin and eosin staining) and hMSC cell staining, and the cell distribution was analyzed. Shown are HE-stained sections on the fifth day after transplantation (FIGS. 34 and 35).

  From FIG. 34, in the mosaic cell mass, the nucleus was clear even in the central hMSC cell, and the hMSC of the entire mosaic cell mass was 100% alive. On the other hand, from FIG. 35, in the cell-only mass, the nucleus concentrated and obscured in the center, the hMSC cells were necrotic, and the cell population alive was 62.7%. In the living body, it was confirmed that hMSC was necrotic at the center in the hMSC cell mass, whereas hMSC was able to survive to the center in the hMSC mosaic cell mass.

  In addition, it was confirmed that blood vessels were formed inside the hMSC mosaic cell cluster on the fifth day. The number of blood vessels was 6 per area. On the other hand, in the hMSC cell mass, no blood vessel formation was observed inside, and the number of blood vessels per area was zero. In the living body, hMSC mosaic cell masses form blood vessels inside, creating an environment suitable for cell survival.

Example 22- (1): Transplantation of Mosaic Cell Mass in Mice Using MMS and hECFC Mosaic Cell Masses Mice are NOD / SCID (Charles River) male, 4 weeks old, 8 weeks old About 12 weeks of age were used. Under anesthesia, the mouse abdomen body hair was removed, a cut was made in the upper abdomen subcutaneously, scissors were inserted from the cut, and the skin was peeled off from the muscle, and then the three types prepared in Examples 20 (1) to (3) The hMSC + hECFC mosaic cell mass was scooped with tweezers, transplanted subcutaneously about 1.5 cm from the incision toward the lower abdomen, and the skin incision was sutured.

Collection of mosaic cell mass Dissection was performed 5 days, 14 days and 28 days after transplantation. The skin of the abdomen was peeled off, and the skin to which the mosaic cell mass adhered was cut into a square size of about 1 square cm. When the mosaic cell mass was also attached to the abdominal muscle, it was collected together with the muscle.

  Tissue sections were prepared for the skin pieces to which the mosaic cell mass was attached and the mosaic cell mass before transplantation. The skin was immersed in 4% paraformaldehyde and formalin fixation was performed. Then, it embedded with paraffin and produced the tissue section | slice of the skin containing a mosaic cell mass. The sections were immunostained with CD31 antibody and DAB coloring method for HE staining (hematoxylin and eosin staining) and hECFC staining, and angiogenesis inside the mosaic cell mass and behavioral state of hMSC and hECFC were analyzed. A HE-stained section on the fifth day after transplantation is shown.

  FIG. 36 is a HE-stained section of the mosaic cell mass produced in Example 20- (1), and FIG. 37 is the HE of the mosaic cell mass produced in Example 20- (2). FIG. 38 is a HE-stained section of the desiccated slice, FIG. 38, which is intended to use the mosaic cell mass produced in Example 20- (3). From any figure, it can be seen that blood vessels are formed inside the mosaic cell mass on the fifth day after transplantation. Example 20- (2) Mosaic cell mass produced by A, Mosaic cell mass produced by Example 20- (3), Mosaic cell mass produced by Example 20- (1) A lot of formation was observed, and it was demonstrated that the cell survival of the mosaic cell mass was clearly better than that of the cell mass in vivo. In addition, it has been proved that blood vessels can be formed inside the mosaic cell mass, and in particular, the ability to form blood vessels is enhanced by the presence of hECFC.

Example 22- (2): Transplantation of Mosaic Cell Mass in Mice Using MMS and hECFC Mosaic Cell Mass Mice Transplanted with NOD / SCID (Charles River) male, 4 weeks old . Under anesthesia, the mouse abdominal body hair was removed, a cut was made in the upper abdomen subcutaneously, scissors were inserted from the cut, and the skin was peeled off from the muscle, followed by two patterns A and B prepared in Example 20 (2). The hMSC + hECFC mosaic cell mass of A and B of Comparative Example 2 and the two patterns of hMSC + hECFC cell mass of Comparative Example 2 were scooped with tweezers, transplanted subcutaneously into the flank about 1.5 cm from the incision, and the skin incision was Sutured.

Collection of Mosaic Cell Mass Dissection was performed 6 days, 14 days and 28 days after transplantation. The skin of the abdomen was peeled off, and the skin to which the mosaic cell mass adhered was cut into a square size of about 1 square cm. When the mosaic cell mass was also attached to the abdominal muscle, it was collected together with the muscle.

Tissue sections were prepared for the skin pieces to which the mosaic cell mass was attached and the mosaic cell mass before transplantation. The skin was immersed in 4% paraformaldehyde and formalin fixation was performed. Then, it embedded with paraffin and produced the tissue section | slice of the skin containing a mosaic cell mass. The sections were immunostained with CD31 antibody and DAB coloring method for HE staining (hematoxylin and eosin staining) and hECFC staining, and angiogenesis inside the mosaic cell mass and behavioral state of hMSC and hECFC were analyzed. A HE-stained section at 14 days after transplantation is shown.
FIG. 39 is a HE-stained section of Example 20- (2) A mosaic cell mass to be used, and FIG. 40 is an HE-stained section of Example 20- (2) B mosaic cell cluster to be used. FIG. From either figure, it can be seen that blood vessels are formed inside the mosaic cell mass on the 14th day after transplantation, but the mosaic cell mass of B in Example 20- (2) is more in the mosaic cell mass of A. More blood vessel formation was seen. In the mosaic cell mass, in addition to the presence of hECFC, it was proved that the higher the number of hECFC, the higher the angiogenic ability. On the other hand, FIG. 41 is a HE-stained section of B using Comparative Example 2. The cell mass of A of Comparative Example 2 could not be collected on the 14th day after transplantation. On the other hand, in the cell mass of B of Comparative Example 2, it was observed that the cell mass became small, blood vessels were not seen, and cells were dead. As a result, even when hECFC was included in the same manner, it was confirmed that a cell mass without a block could not form blood vessels and cells died.

Claims (24)

A cell structure for cell transplantation comprising a polymer block having biocompatibility and at least one kind of cell, wherein a plurality of the polymer blocks are arranged in a gap between the plurality of cells. ,
The polymer block has a size of 20 μm or more and 150 μm or less,
The shape of the polymer block is irregular,
A cell structure for cell transplantation, wherein the ratio of the polymer block to the cells is 0.0000001 μg or more and 1.0 μg or less per cell.   The cell structure for cell transplantation according to claim 1, which has a thickness or diameter of 400 µm or more and 3 cm or less.   The cell structure for cell transplantation according to claim 1, wherein the thickness or diameter is 500 µm or more and 2 cm or less.   The cell structure for cell transplantation according to claim 2 or 3, wherein the thickness or diameter is 720 µm or more and 1 cm or less.   The cell structure for cell transplantation according to any one of claims 1 to 4, which is produced by incubating a mixture of a polymer block having biocompatibility and a cell-containing culture solution.   The cell structure for cell transplantation according to any one of claims 1 to 5, wherein the polymer having biocompatibility is a biodegradable material.   The polymer having bioaffinity is a polypeptide, polylactic acid, polyglycolic acid, PLGA, hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, or chitosan. The cell structure for cell transplant according to any one of 1 to 6.   The polymer having bioaffinity is gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, or retronectin according to any one of claims 1 to 7. Cell structure for cell transplantation.   The cell structure for cell transplantation according to any one of claims 1 to 8, wherein the biocompatible polymer is crosslinked.   The cell structure for cell transplantation according to claim 9, wherein the crosslinking is performed with aldehydes, a condensing agent, or an enzyme.   The cell structure for cell transplantation according to any one of claims 1 to 10, wherein the polymer having biocompatibility is a recombinant peptide.   The cell structure for cell transplantation according to claim 11, wherein the polymer having bioaffinity has two or more cell adhesion signals in one molecule. Recombinant peptide is
Formula: A-[(Gly-XY) _n ] _m -B
(In the formula, A represents an arbitrary amino acid or amino acid sequence, B represents an arbitrary amino acid or amino acid sequence, n Xs independently represent any of the amino acids, and n Ys each independently represent an amino acid. N represents an integer of 3 to 100, m represents an integer of 2 to 10. Note that n Gly-XY may be the same or different. Item 12. A cell structure for cell transplantation according to Item 11. Recombinant peptide is
Formula: Gly-Ala-Pro-[(Gly-XY) ₆₃ ] ₃ -Gly
(In the formula, 63 X's each independently represent any amino acid, and 63 Y's each independently represent any amino acid. The 63 Gly-XYs may be the same or different. The cell structure for cell transplantation according to claim 11 or 13, represented by   The recombinant peptide has (1) an amino acid sequence described in SEQ ID NO: 1 or (2) an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility. Item 15. The cell structure for cell transplant according to any one of Items 11 to 14.   The cell structure for cell transplantation according to any one of claims 1 to 15, comprising an angiogenic factor.   The cell structure for cell transplantation according to any one of claims 1 to 16, wherein the cell is a cell selected from the group consisting of a universal cell, a somatic stem cell, a progenitor cell, and a mature cell.   The cell structure for cell transplantation according to claim 17, wherein the cells include non-vascular cells.   The cell structure for cell transplantation according to claim 17, wherein the cells are only non-vascular cells.   19. The cell structure for cell transplantation according to claim 18, wherein the cells are two or more types and include both non-vascular cells and vascular cells.   21. The cell structure for cell transplantation according to claim 20, wherein the cell structure has a region where the area of cells in the central vascular system is larger than the area of cells in the peripheral vascular system.   The cell structure for cell transplantation according to claim 21, wherein a ratio of the vascular cells in the central part is 60% to 100% with respect to the total area of vascular cells. 23. The cell transplantation cell according to any one of claims 20 to 22, wherein the cell structure has a region where the cell density of the vascular system in the central part is 1.0 × 10 ⁻⁴ cells / μm ³ or more. Structure.   24. A cell structure for cell transplantation, which is vascularized using the cell structure for cell transplantation according to any one of claims 20 to 23.