WO2011108517A1 - 生体親和性を有する高分子ブロックと細胞からなる細胞構造体 - Google Patents
生体親和性を有する高分子ブロックと細胞からなる細胞構造体 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/76—Agarose, agar-agar
Definitions
- the present invention relates to a cell structure comprising a polymer block having biocompatibility and a cell, wherein a plurality of the polymer blocks are arranged in a mosaic pattern in the gaps between the plurality of cells, and It relates to the manufacturing method.
- Regenerative medicine is being put to practical use to measure 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.
- the cells to be transplanted are mainly transplanted in the form of a thin sheet or transplanted in a suspension state, so that a tissue having a sufficient thickness cannot be provided.
- a living tissue originally has a thickness, and thus has a thickness. Therefore, the body tissue enables a muscle force to beat the heart, and enables a smooth movement in the articular cartilage.
- tissue regeneration using cells it is considered that a major problem is that a tissue having a thickness cannot be provided.
- Non-Patent Documents 1 to 6 Such a cell sheet manufacturing technique is expected to be useful for regeneration of myocardial tissue.
- a vascular network cannot be formed with conventional cell sheets, it has been difficult to regenerate a sufficiently thick tissue (Non-patent Documents 5 and 7).
- 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.
- 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).
- inventions are methods in which a sheet with cultured osteoblasts attached is placed in the body, and cortical bone is formed from osteoblasts by membranous ossification in the body.
- a sheet using an osteoblast layer could not provide a regenerated sheet having a cell layer thickness exceeding 100 ⁇ m.
- Patent Document 3 a sheet having a thickness of 200 ⁇ m or more can be formed by developing and optimizing the culture technique, but only a thickness of about 210 ⁇ m can be actually provided.
- Non-Patent Document 9 a gel-embedded culture using collagen has been devised.
- 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.
- 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.
- Patent Document 4 states that three-dimensional culture is possible by connecting cells with inorganic ceramic beads.
- 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.
- cells are adhered to particles of 150 to 460 ⁇ m, and a thick nonwoven fabric (1 cm) of PLLA is laminated around it to increase the apparent thickness.
- the inclusion layer is only a few tens of ⁇ m 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.
- An object of the present invention is to provide a cell structure having a thickness sufficient for tissue regeneration and in which cells uniformly exist in the structure. Furthermore, an object of the present invention is to provide a cell structure that can be produced without using cells other than the target cells. Furthermore, an object of the present invention is to provide a cell structure in which cell three-dimensional structures can be naturally fused.
- the present inventors have arranged a three-dimensional arrangement of a cell three-dimensional structure from the outside by arranging three-dimensionally a polymer block having a biocompatibility (a lump made of a polymer material having biocompatibility) and cells in a mosaic.
- a biocompatibility a lump made of a polymer material having biocompatibility
- cells in a mosaic.
- natural fusion between the three-dimensional cell structures is possible by the action of the cells present on the outer periphery of the three-dimensional cell structure.
- the present invention has been completed based on these findings.
- a cell structure comprising a polymer block having biocompatibility and a cell, wherein a plurality of the polymer blocks are disposed in a gap between the plurality of cells.
- [5] The cell structure according to [4], wherein the thickness or diameter is 720 ⁇ m or more and 1 cm or less.
- [6] The cell structure according to any one of [1] to [5], wherein the ratio of the polymer block to the cells is 0.0000001 ⁇ g to 1.0 ⁇ g per cell.
- [8] The cell structure according to any one of [1] to [7], wherein the biocompatible polymer is a biodegradable material.
- the biocompatible polymer is a polypeptide, polylactic acid, polyglycolic acid, PLGA, hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, or chitosan.
- the cell structure according to any one of [1] to [8].
- the polymer having bioaffinity is gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, or retronectin, any of [1] to [9]
- a cell structure according to claim 1. [11] The cell structure according to any one of [1] to [10], wherein the biocompatible polymer is crosslinked. [12] The cell structure according to [11], wherein the crosslinking is performed with an aldehyde, a condensing agent, or an enzyme. [13] The cell structure according to any one of [1] to [12], wherein the biocompatible polymer is genetically modified gelatin. [14] The cell structure according to [13], wherein the polymer having bioaffinity has two or more cell adhesion signals in one molecule.
- the genetically modified gelatin 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 from each other. [13] The cell structure according to [13].
- the genetically modified gelatin is (1) an amino acid sequence described in SEQ ID NO: 1, or (2) an amino acid sequence having a biocompatibility having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1.
- the cell structure according to any one of [13] to [16].
- the method according to [18] wherein the medium is exchanged in the step of incubating the mixture of the biocompatible polymer block and the cell-containing culture solution.
- the medium is replaced with a differentiation medium or a growth medium in the step of incubating the mixture of the biocompatible polymer block and the cell-containing culture medium.
- a plurality of polymer blocks having biocompatibility and a plurality of cells, and a part or all of a plurality of gaps formed by the plurality of cells include one or a plurality of the high blocks
- a method for producing a cell structure comprising a step of fusing a plurality of cell structures on which molecular blocks are arranged.
- each cell structure before fusion is 10 ⁇ m or more and 1 cm or less, and the thickness or diameter after fusion is 400 ⁇ m or more and 3 cm or less, according to [25] or [26] A method for producing a cell structure.
- a plurality of first polymer blocks having biocompatibility and a plurality of cells, and one or more in some or all of the plurality of gaps formed by the plurality of cells A method for producing a cell structure, further comprising the step of adding and incubating a second polymer block to the cell structure in which the polymer block is disposed.
- [31] The method for producing a cell structure according to any one of [28] to [30], wherein the thickness or diameter after adding the second polymer block and incubating is 400 ⁇ m or more and 3 cm or less.
- [32] A cell structure produced by the method for producing a cell structure according to any one of [25] to [31].
- [33] The cell structure according to [32], which is used for cell transplantation, cell culture, or toxicity evaluation.
- the cell structure of the present invention has a thickness sufficient for tissue regeneration, and the cells are uniformly present in the structure.
- the cell structure of the present invention can be produced without using cells other than the target cell. Furthermore, in the cell structure of the present invention, the cell structures can be naturally fused.
- the cell structure of the present invention is useful in regenerative medicine for measuring the regeneration of biological tissues / organs that have suffered from dysfunction or malfunction.
- FIG. 1 shows a stereoscopic microscope photograph of Day 7 (cartilage differentiation medium) of a mosaic cell mass produced with a recombinant gelatin ⁇ block.
- FIG. 2 shows a stereoscopic microscope photograph of Day 7 (cartilage differentiation medium) of a mosaic cell mass produced with a natural gelatin ⁇ block.
- FIG. 3 shows a photograph of a section of a mosaic cell mass using a recombinant gelatin ⁇ block (HE staining ⁇ 5 times).
- FIG. 4 shows a photograph of a section of a mosaic cell mass using recombinant gelatin ⁇ block (HE staining ⁇ 10 times).
- FIG. 1 shows a stereoscopic microscope photograph of Day 7 (cartilage differentiation medium) of a mosaic cell mass produced with a recombinant gelatin ⁇ block.
- FIG. 2 shows a stereoscopic microscope photograph of Day 7 (cartilage differentiation medium) of a mosaic cell mass produced with a natural gelatin ⁇
- FIG. 5 shows a photograph of a section of a mosaic cell mass using a recombinant gelatin ⁇ 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 / 3 ⁇ r 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 times) using a recombinant gelatin ⁇ block.
- FIG. 17 shows a section of a mosaic cell mass (Day 7 (under growth medium), ⁇ 10 times) using a recombinant gelatin ⁇ block.
- FIG. 18 shows a HE section photograph ( ⁇ 5) of Day 21 (recombinant gelatin block added under growth medium added) with increased volume.
- FIG. 19 shows Day21 with increased volume (recombinant gelatin block added under growth medium)
- FIG. 20 shows HE section photographs ( ⁇ 5 times, ⁇ 20 times) of Day 21 (with a recombinant gelatin 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 PLGA ⁇ block.
- FIG. 25 shows a state in which a mosaic cell mass using cardiomyocytes and a recombinant gelatin ⁇ block pulsates synchronously as a whole.
- FIG. 26 shows a fluorescence micrograph and a micrograph of a mosaic cell mass using GFP-expressing HUVEC and a recombinant gelatin ⁇ block. (50,000 cells + 0.03 mg mosaic cell mass and 300,000 cells + 0.2 mg mosaic cell mass)
- the cell structure of the present invention comprises a polymer block having bioaffinity and cells, and a plurality of the polymer blocks are arranged in the gaps between the plurality of cells. is there.
- 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.
- 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.
- a plurality of the polymer blocks are disposed in the gaps between the plurality of cells.
- the “gap between cells” is closed by the cells that are constituted. It is not necessary for the space to be 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 ⁇ m or more and 1000 ⁇ m or less, preferably 10 ⁇ m or more and 100 ⁇ m or less, and more preferably 10 ⁇ m or more and 50 ⁇ m or less.
- 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.
- the non-biodegradable material include materials made of at least one selected from PTFE, polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, stainless steel, titanium, silicone, and MPC.
- biodegradable material specifically, at least selected from polypeptide, polylactic acid, polyglycolic acid, PLGA, hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, chitosan
- 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
- 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
- RGD sequence amino acid sequence
- LDV sequence cell adhesion sequence
- REDV sequence YIGSR sequence
- PDSGR sequence RYVVLPR sequence
- LGTIPPG sequence RNIAEIIKDI sequence
- IKVAV sequence IKVAV sequence
- LRE sequence DGEA sequence
- HAV sequence HAV sequence
- polypeptide is not particularly limited as long as it has biocompatibility.
- gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, and retronectin are most preferable.
- Gelatin, collagen and atelocollagen are preferred.
- the gelatin for use in the present invention is preferably natural gelatin or genetically modified gelatin. More preferred is genetically modified gelatin.
- natural gelatin means gelatin made from naturally derived collagen. The genetically modified gelatin 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.
- methane (CH 4 ) is the source of all organic compounds, and all the other compounds are all methane derivatives, with certain numbers set for their carbon number, substituents, transformations, rings, etc. Then, the score is added to obtain an organic value (OV) and an inorganic value (IV), and these values are plotted on a diagram with the organic value on the X axis and the inorganic value on the Y axis. It is going.
- the IOB in the organic conceptual diagram 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)”.
- hydrophilicity / hydrophobicity is represented by a “1 / IOB” value obtained by taking the reciprocal of IOB.
- 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.
- 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.
- the biocompatible polymer material used in the present invention may be cross-linked or non-cross-linked, but is preferably cross-linked.
- 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.
- photocrosslinking examples include photoirradiation to a polymer into which a photoreactive group has been introduced, or light irradiation in the presence of a photosensitizer.
- the photoreactive group examples include a cinnamyl group, a coumarin group, a dithiocarbamyl group, a xanthene dye, and camphorquinone.
- the enzyme 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.
- 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.
- Human-derived blood coagulation factors such as guinea pig liver-derived transglutaminase, goat-derived transglutaminase, and rabbit-derived transglutaminase manufactured by Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign International, etc. Etc.
- Human-derived blood coagulation factors such as guinea pig liver-derived transglutaminase, goat-derived transglutaminase, and rabbit-derived transglutaminase manufactured by Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign International, etc. 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.
- the mixing temperature for treating the polymer material 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 ° C to 30 ° C, more preferably 3 ° C to 25 ° C, still more preferably 3 ° C to 15 ° C, still more preferably 3 ° C to 10 ° C, and particularly preferably 3 ° C to 7 ° C.
- 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 ° C to 40 ° C, more preferably 3 ° C to 25 ° C, more preferably 3 ° C to 15 ° C, still more preferably 3 ° C to 10 ° C, and particularly preferably 3 ° C to 7 ° C.
- 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 further preferably 120 ° C to 200 ° C.
- the genetically modified gelatin according to the present invention means a polypeptide or protein-like substance having an amino acid sequence similar to gelatin produced by a genetic recombination technique.
- the genetically modified gelatin that can be used in the present invention is preferably one having a repeating sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents any one of amino acids). Gly-XY may be the same or different.
- two or more sequences of cell adhesion signals are contained in one molecule.
- a genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used, for example, those described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041, etc. However, it is not limited to these.
- Preferred as the genetically modified gelatin used in the present invention is the genetically modified gelatin of the following embodiment.
- the genetically modified gelatin used in the present invention is excellent in biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived.
- the genetically modified gelatin used in the present invention is more uniform than natural ones and the sequence is determined, the strength and degradability can be precisely designed with less blur due to cross-linking described later. It is.
- the molecular weight of the genetically modified gelatin is preferably 2 to 100 KDa. More preferably, it is 2.5 to 95 KDa. More preferably, it is 5 to KDa. Most preferably, it is 10 KDa or more and 90 KDa or less.
- the genetically modified gelatin has a repeating sequence represented by Gly-XY, which is characteristic of collagen.
- the plurality of Gly-X-Ys may be the same or different.
- Gly-XY Gly represents glycine
- 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.
- 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.
- General gelatin has 1: 1 polar amino acids, both charged and uncharged.
- the polar amino acid specifically refers to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine, arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. , Threonine, tyrosine.
- the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituent amino acids.
- 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.
- the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology”, Vol. 9, No. 7 (1990), page 527, published by Nagai Publishing Co., Ltd.).
- the genetically modified gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule.
- Specific sequences include RGD sequences, LDV sequences, REDV sequences, YIGSR sequences, PDSGR sequences, RYVVLPR sequences, LGITIPG sequences, RNIAEIIKDI sequences, which are expressed in one-letter amino acid notation in that many types of cells adhere.
- IKVAV 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.
- RGD sequences an ERGD sequence is preferred.
- the number of amino acids between RGDs is not uniform between 0 and 100, preferably between 25 and 60.
- the content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 per protein molecule from the viewpoint of cell adhesion and proliferation. Most preferably, it is 12.
- the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%.
- each stretch of 350 amino acids has at least 1 stretch.
- it contains two RGD motifs.
- 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 genetically modified gelatin 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.
- the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains one RGD sequence.
- it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less.
- the genetically modified gelatin may be partially hydrolyzed.
- the genetically modified gelatin 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.
- 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.
- the collagen origin is preferably human, cow, pig, mouse, rat. More preferably, it is a human.
- the isoelectric point of the genetically modified gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.
- the genetically modified gelatin is not deaminated.
- the genetically modified gelatin has no telopeptide.
- the genetically modified gelatin is a substantially pure collagen material prepared with a nucleic acid encoding natural collagen.
- the genetically modified gelatin 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 genetically modified gelatin having
- the genetically modified gelatin used in the present invention can be produced by a genetic recombination technique known to those skilled in the art, for example, 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 gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is then introduced into a suitable host to produce a transformant. To do. By culturing the obtained transformant in an appropriate medium, genetically modified gelatin is produced.
- the genetically modified gelatin used in the present invention is prepared by recovering the genetically modified gelatin produced from the culture. be able to.
- a block (lumb) made of the polymer having bioaffinity described above 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 ⁇ m or more and 700 ⁇ m or less, more preferably 10 ⁇ m or more and 700 ⁇ m or less, further preferably 10 ⁇ m or more and 300 ⁇ m or less, further preferably 20 ⁇ m or more and 150 ⁇ m or less, and particularly preferably It is 25 ⁇ m or more and 106 ⁇ m or less.
- the polymer block may be in the form of a long string of 700 ⁇ m 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.
- the cell used in the present invention can be appropriately selected according to the purpose of the cell structure of the present invention, and the type thereof is not particularly limited. Further, the cells may be used alone or in combination of a plurality of types depending on the purpose of use of the cell structure.
- the cell to be used is preferably an animal cell, more preferably a vertebrate cell, particularly preferably a human cell.
- Vertebrate-derived cells may be any of universal cells, somatic stem cells, progenitor cells, or mature cells.
- ES cells, GS cells, or iPS cells can be used as the universal cells.
- 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.
- MSC mesenchymal stem cells
- 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.
- human-derived cells examples 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.
- the origin of the cell may be either an autologous cell or an allogeneic cell. When a plurality of types of cells are used in combination, for example, vascular cells and other cells are listed.
- Vascular cells include vascular endothelial cells, vascular endothelial precursor cells, hematopoietic stem cells, and the like. By combining vascular cells and other cells, blood vessels can be induced in the cell structure of the present invention, and nutrition, oxygen, and the like can be supplied.
- the thickness or diameter of the cell structure of the present invention can be set to a desired thickness by the method described later in this specification, but the lower limit is preferably 215 ⁇ m or more, more preferably 400 ⁇ m or more, and 730 ⁇ m. The above is most preferable.
- 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 ⁇ m to 3 cm, more preferably 500 ⁇ m to 2 cm, and still more preferably 720 ⁇ m to 1 cm.
- a cell structure FIG.
- the cell structure 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.
- the “thickness or diameter of the cell structure” in this specification indicates the following. When a certain point A in the cell structure is selected, the length of the line segment that divides the cell structure so that the distance from the outside of the cell structure becomes the shortest in a straight line passing through the point A is shown. Minute A. A point A having the longest line segment A is selected in the cell structure, and the length of the line segment A at that time is defined as the “thickness or diameter of the cell structure”.
- the cell structure of the present invention can have a sufficient thickness, and since cells can exist uniformly, it can be suitably used for cell transplantation, cell culture, toxicity evaluation and the like.
- the thickness or diameter of the cell structure of the present invention is preferably in the above range.
- the range of the thickness or diameter of the cell structure is preferably 10 ⁇ m or more and 1 cm or less, more preferably 10 ⁇ m or more and 2000 ⁇ m or less, still more preferably 15 ⁇ m or more and 1500 ⁇ m or less, and most preferably 20 ⁇ m or more and 1300 ⁇ m or less.
- the ratio of cells to polymer blocks is not particularly limited, but the ratio of polymer blocks per cell is preferably 0.0000001 ⁇ g or more and 1.0 ⁇ g or less, more preferably. Is 0.000001 ⁇ g to 0.1 ⁇ g, more preferably 0.00001 ⁇ g to 0.01 ⁇ g, and most preferably 0.00002 ⁇ g to 0.006 ⁇ g. From this range, the cells can be present more uniformly. In addition, by setting the lower limit to the above range, the effect of the 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.
- the component in the polymer block is not particularly limited, and examples thereof include components contained in the medium described later.
- 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.
- 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.
- 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.
- 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 used is preferably a container made of a low-cell adhesive material or a non-cell-adhesive material, more preferably a container made of polystyrene, polypropylene, polyethylene, glass, polycarbonate, or polyethylene terephthalate.
- the shape of the bottom surface of the container is preferably a flat bottom type, a U shape, or a V shape.
- 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.
- the cell structure in the step of incubating a mixture of a biocompatible polymer block and a cell-containing culture solution, can be increased in volume by exchanging the medium with a differentiation medium or a growth medium.
- a cell structure having a desired size is obtained by further adding the biocompatible polymer block.
- 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.
- the thickness or diameter of each cell structure before the fusion is 10 ⁇ m or more and 1 cm or less, and the thickness or diameter after the fusion is 400 ⁇ m or more and 3 cm or less.
- the thickness or diameter of each cell structure before fusion is more preferably 10 ⁇ m or more and 2000 ⁇ m or less, further preferably 15 ⁇ m or more and 1500 ⁇ m or less, and most preferably 20 ⁇ m or more and 1300 ⁇ m or less.
- the range of the thickness or the diameter is more preferably 500 ⁇ m or more and 2 cm or less, and further preferably 720 ⁇ m 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.
- a suitable range such as “block ratio” is the same as the preferred range relating to the cell structure of the present invention.
- the cell structures to be fused are preferably placed at a distance of 0 to 50 ⁇ m, more preferably 0 to 20 ⁇ m, and still more preferably 0 to 5 ⁇ m.
- 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 ⁇ m to 700 ⁇ m, more preferably 10 ⁇ m to 700 ⁇ m, still more preferably 10 ⁇ m to 300 ⁇ m, and further preferably 20 ⁇ m to 150 ⁇ m. Or less, particularly preferably 25 ⁇ m or more and 106 ⁇ m or less.
- the size of the second polymer block according to the present invention is also preferably 1 ⁇ m to 700 ⁇ m, more preferably 10 ⁇ m to 700 ⁇ m, still more preferably 10 ⁇ m to 300 ⁇ m, and further preferably 20 ⁇ m to 150 ⁇ m. Or less, particularly preferably 25 ⁇ m or more and 106 ⁇ m 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 ⁇ m or more and 3 cm or less, more preferably 500 ⁇ m or more and 2 cm or less, and further preferably 720 ⁇ m 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.
- the cell structure produced by the method for producing a cell structure of the present invention can form a desired shape and size, can have a sufficient thickness, and cells can exist uniformly. It can be suitably used for cell transplantation, cell culture and toxicity evaluation.
- Example 1 Genetically modified gelatin CBE3 described below was prepared as a genetically modified gelatin (recombinant gelatin) (described in WO2008-103041).
- 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 Preparation of Recombinant Gelatin ⁇ Block An amorphous ⁇ block was prepared using recombinant gelatin CBE3 as a base block. Dissolve 1000 mg of recombinant gelatin in 9448 ⁇ L of ultrapure water, add 152 ⁇ L of 1N HCl, add 400 ⁇ L 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.
- 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 washed cross-linked gelatin gel 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 a 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 gelatin ⁇ blocks of 25 to 53 ⁇ m and 53 to 106 ⁇ m.
- Example 3 Preparation of Natural Gelatin ⁇ Block An amorphous ⁇ 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 ⁇ L of ultrapure water, add 152 ⁇ L of 1N HCl, add 400 ⁇ L 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.
- natural gelatin Nippi, Nippi Gelatin High Grade Gelatin APAT
- 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 washed cross-linked gelatin gel 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 a 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 ⁇ blocks of 25 to 53 ⁇ m and 53 to 106 ⁇ m.
- Example 4 Production of Mosaic Cell Mass Using Recombinant Gelatin ⁇ Block Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 500,000 cells / mL in a growth medium (Takara Bio: MSCGM-CD TM BulletKit TM ). After adding the recombinant gelatin ⁇ block prepared in Example 2 to 1.0 mg / mL, 100 ⁇ L was seeded on a Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom U-shaped) and allowed to stand for 18 hours.
- hMSC Human bone marrow-derived mesenchymal stem cells
- Example 5 Production of Mosaic Cell Mass Using Natural Gelatin ⁇ Block Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 500,000 cells / mL with a growth medium (Takara Bio: MSCGM-CD TM BulletKit TM ). After adding the natural gelatin ⁇ block prepared in Example 3 to 1.0 mg / mL, 100 ⁇ L 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 ⁇ block and hMSC cells was prepared (0.002 ⁇ g polymer block per cell).
- the medium was replaced with cartilage differentiation medium (Takara Bio: hMSC Differentiation BulletKit TM , Chondrogenic, TGF- ⁇ 3) (200 ⁇ L).
- cartilage differentiation medium (Takara Bio: hMSC Differentiation BulletKit TM , Chondrogenic, TGF- ⁇ 3) (200 ⁇ L).
- this mosaic cell mass is produced in a U-shaped plate, it is produced in a spherical shape.
- hMSC Human bone marrow-derived mesenchymal stem cells
- Example 6 Sample analysis A tissue section was prepared for the mosaic cell mass using the recombinant gelatin ⁇ block prepared in Example 4. To the mosaic cell mass in the medium produced in Example 4, after removing the medium, 200 ⁇ L 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 ⁇ block was analyzed. The results are shown in FIG. 3, FIG. 4 and FIG.
- 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.
- 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.
- 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 Gelatin ⁇ Block Human bone marrow-derived mesenchymal stem cells (hMSC) were grown in growth medium (Takara Bio: MSCGM-CD TM BulletKit TM ) at 500,000 cells / After adjusting to mL and adding the recombinant gelatin ⁇ block prepared in Example 2 to 1.0 mg / mL, 100 ⁇ L 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 ⁇ g polymer block per cell).
- 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 ⁇ m 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 gelatin ⁇ block prepared in Example 2 was added to the growth medium (Takara Bio: MSCGM-CD TM BulletKit) when the medium was replaced. TM ) was added in suspension. Thereafter, 0.1 mg of recombinant gelatin ⁇ block was added in accordance with the medium exchange of Day 7, 10, 14, 17, 21.
- FIGS. The tissue sections (HE staining) at this time are shown in FIGS. It can be seen that cells and recombinant gelatin ⁇ 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.
- Example 10 Volume increase of mosaic cell mass (under cartilage differentiation medium)
- 0.1 mg recombinant gelatin ⁇ block (0.1 mg) prepared in Example 2 was replaced with cartilage differentiation medium (Takara Bio: hMSC Differentiation BulletKit TM , Chondrogenic, TGF- ⁇ 3) was added in suspension. Thereafter, 0.1 mg of recombinant gelatin ⁇ 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 / 3 ⁇ r 3 ),
- the change with time for each is shown in FIGS.
- it was finally formed into a spherical shape with an average diameter ( thickness) of 2.05 mm on Day 21.
- a mosaic cell mass having a size of at least 2.05 mm can be produced.
- the size can be increased by continuing the volume increase by this method.
- tissue sections at this time are shown in FIGS. It can be seen that cells and recombinant gelatin ⁇ 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 ⁇ m.
- 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 gelatin, 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.
- Dimethylmethylene blue dye Frndale et al., Improved quantitation and sulphated glycosaminoglycans by use of dimethylmethylene blue.
- the results of quantifying the amount of GAG over time are shown in the graph of FIG.
- the amount of glycosaminoglycan (GAG) produced was low, whereas the mosaic cell mass produced with the natural gelatin ⁇ block and the recombinant gelatin ⁇ block were produced.
- the amount of GAG produced was extremely high for the mosaic cell mass produced by putting 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.
- the amount of GAG produced was significantly higher in the mosaic cell mass prepared by inserting the recombinant gelatin ⁇ block than in the mosaic cell mass prepared by adding the natural gelatin ⁇ block.
- the mosaic cell mass produced with the recombinant gelatin ⁇ block can maintain higher cell activity and substrate production activity than the natural gelatin ⁇ block. It has been shown that this can be achieved by using recombinant gelatin.
- 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.
- CellTiter-Glo Promega
- the mosaic cell mass produced in Example 4 and Example 5 hMSC cell + recombinant gelatin, hMSC cell + natural gelatin
- the ATP content in each mosaic cell mass was quantified using CellTiter-Glo.
- the mosaic cell mass produced using the gelatin ⁇ block had a significantly higher ATP production / retention amount than the cell mass produced only with the cells (p ⁇ 0.01). This is because when the gelatin block is fitted in a mosaic shape, a nutrient supply route into the mosaic cell mass by the gelatin 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. Furthermore, it was found that the amount of ATP produced / retained was significantly higher in the mosaic cell mass prepared by inserting the recombinant gelatin ⁇ block than in the mosaic cell mass prepared by adding the natural gelatin ⁇ block.
- the mosaic cell mass produced by inserting the recombinant gelatin ⁇ block had higher cell survival than the natural gelatin ⁇ block, and the cells were alive inside. It has been shown that improved cell survival, which was not possible with natural gelatin, can be achieved by using recombinant gelatin.
- Example 13 Preparation of PLGA ⁇ 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 ⁇ blocks of 25 to 53 ⁇ m and 53 to 106 ⁇ m. 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 TM BulletKit TM ) After adding the PLGA ⁇ 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 ⁇ L 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 ⁇ g per cell).
- Example 15 Preparation of agarose ⁇ 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 agarose ⁇ blocks of 25 to 53 ⁇ m and 53 to 106 ⁇ m. IOB value: 3.18
- Example 16 Production of Mosaic Cell Mass Using Agarose
- hMSC human bone marrow-derived mesenchymal stem cell
- a growth medium Takara Bio: MSCGM-CD TM BulletKit TM
- 15 was added (prepared by shaking the conditions so that the final concentration was 0.1 mg / mL and 1.0 mg / mL), 100 ⁇ L 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 ⁇ g polymer block per cell).
- the medium was increased to 200 ⁇ L, and the medium was changed every three days for cultivation.
- 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).
- 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.
- 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).
- the cells were adjusted to 1.5 million cells / mL, the recombinant gelatin ⁇ block prepared in Example 2 was added to 1.0 mg / mL, and then 100 ⁇ L and 200 ⁇ L were added to Sumilon Celtite X96U plate. What was seeded on (Sumitomo Bakelite, U-shaped bottom) was also produced. All were allowed to stand for 18 hours to prepare a mosaic cell mass composed of a recombinant gelatin ⁇ block having a diameter of about 1 to 2 mm and GFP-HUVEC cells. Medium exchange was performed at Day 3, 7, 10, 14, 17, 21.
- FIG. 26 shows micrographs and fluorescence micrographs of a 50,000 cells + 0.03 mg mosaic cell mass and a 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.
- 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.
- it has been shown that it can be formed using various polymer blocks such as a recombinant gelatin block, an animal gelatin block, a PLGA block, and an agarose block.
- the three-dimensional cell structure (mosaic cell mass) of the present invention can be formed in various cell types and various polymer block types.
Abstract
Description
間葉系幹細胞をシート状に培養した培養細胞シートと生分解性物質をシート状に形成した生分解シートとを積層してなる骨再生シート(特許文献1)が提案されている。また、多孔質シート上に間葉系細胞から分化させた間葉系組織前駆体細胞と細胞外基質とが付着している間葉系組織再生誘導用シートもある(特許文献2)。これらの発明は培養骨芽細胞の付着したシートを体内に入れ、体内での膜性骨化によって骨芽細胞から皮質骨を形成させる方法である。しかしながら、骨芽細胞様細胞は積層して培養することができないという問題があるため骨芽細胞層を用いたシートでは細胞層の厚さが100μmを超える再生シートを提供できなかった。その後、特許文献3では、培養手法の開発・最適化によって、200μm以上の厚さのシートを形成できるとあるが、実際のところは210μm程度までの厚みしか提供できていない。
〔1〕 生体親和性を有する高分子ブロックと細胞とを含み、該複数個の細胞間の隙間に複数個の該高分子ブロックが配置されている、細胞構造体。
〔2〕 該高分子ブロックの大きさが1μm以上700μm以下である、〔1〕に記載の細胞構造体。
〔3〕 該高分子ブロックの大きさが10μm以上300μm以下である〔2〕に記載の細胞構造体。
〔4〕 厚さ又は直径が400μm以上3cm以下である、〔1〕~〔3〕のいずれかに記載の細胞構造体。
〔6〕 前記高分子ブロックと前記細胞との比率が、細胞1個当り0.0000001μg以上1.0μg以下である〔1〕~〔5〕のいずれかに記載の細胞構造体。
〔7〕 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートすることによって製造される、〔1〕から〔6〕の何れかに記載の細胞構造体。
〔8〕 生体親和性を有する高分子が、生分解性材料である、〔1〕から〔7〕の何れかに記載の細胞構造体。
〔9〕 生体親和性を有する高分子が、ポリペプチド、ポリ乳酸、ポリグリコール酸、PLGA、ヒアルロン酸、グリコサミノグリカン、プロテオグリカン、コンドロイチン、セルロース、アガロース、カルボキシメチルセルロース、キチン、又はキトサンである、〔1〕から〔8〕の何れかに記載の細胞構造体。
〔11〕 生体親和性を有する高分子が架橋されている、〔1〕から〔10〕の何れかに記載の細胞構造体。
〔12〕 架橋がアルデヒド類、縮合剤、又は酵素により施される、〔11〕に記載の細胞構造体。
〔13〕 生体親和性を有する高分子が、遺伝子組み換えゼラチンである、〔1〕から〔12〕の何れかに記載の細胞構造体。
〔14〕 前記生体親和性を有する高分子が、細胞接着性シグナルを一分子中に2以上有する〔13〕に記載の細胞構造体。
式:A-[(Gly-X-Y)n]m-B
(式中、Aは任意のアミノ酸又はアミノ酸配列を示し、Bは任意のアミノ酸又はアミノ酸配列を示し、n個のXはそれぞれ独立にアミノ酸の何れかを示し、n個のYはそれぞれ独立にアミノ酸の何れかを示し、nは3~100の整数を示し、mは2~10の整数を示す。なお、n個のGly-X-Yはそれぞれ同一でも異なっていてもよい。)で示される、〔13〕に記載の細胞構造体。
式:Gly-Ala-Pro-[(Gly-X-Y)63]3-Gly
(式中、63個のXはそれぞれ独立にアミノ酸の何れかを示し、63個のYはそれぞれ独立にアミノ酸の何れかを示す。なお、63個のGly-X-Yはそれぞれ同一でも異なっていてもよい。)で示される、〔13〕又は〔15〕に記載の細胞構造体。
〔18〕 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程を含む、〔1〕から〔17〕の何れかに記載の細胞構造体の製造方法。
〔19〕 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程において培地交換を行う、〔18〕に記載の方法。
〔20〕 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程において、培地を分化培地又は増殖培地に交換する、〔19〕に記載の方法。
〔22〕 〔18〕から〔21〕の何れかに記載の方法により製造される、細胞構造体。
〔23〕 〔1〕から〔17〕の何れかに記載の細胞構造体の複数個を融合することによって得られる、細胞構造体。
〔24〕 〔1〕から〔17〕の何れかに記載の細胞構造体の複数個を融合する工程を含む、〔23〕に記載の細胞構造体の製造方法。
〔26〕 前記高分子ブロックの大きさが1μm以上700μm以下である、〔25〕に記載の細胞構造体の製造方法。
〔27〕 前記融合前の各細胞構造体の厚さ又は直径が10μm以上1cm以下であり、前記融合後の厚さ又は直径が400μm以上3cm以下である、〔25〕または〔26〕に記載の細胞構造体の製造方法。
〔29〕 前記第一の高分子ブロックの大きさが1μm以上700μm以下である、〔28〕に記載の細胞構造体の製造方法。
〔30〕 前記第二の高分子ブロックの大きさが1μm以上700μm以下である、〔28〕または〔29〕に記載の細胞構造体の製造方法。
〔32〕 〔25〕~〔31〕のいずれかに記載の細胞構造体の製造方法により製造される、細胞構造体。
〔33〕 細胞移植、細胞培養または毒性評価用途に使用する、〔32〕に記載の細胞構造体。
本発明の細胞構造体は、生体親和性を有する高分子ブロックと細胞とを含み、該複数個の細胞間の隙間に複数個の該高分子ブロックが配置されていることを特徴とするものである。実施態様として、生体親和性を有する、複数個の高分子ブロックと、複数個の細胞とを含み、該複数の細胞により形成される複数個の隙間の一部または全部に、一または複数個の前記高分子ブロックが配置されている細胞構造体が挙げられる。
(1-1)高分子材料
本発明で用いる生体親和性を有する高分子は、生体に親和性を有するものであれば、生体内で分解されるか否かは特に限定されないが、生分解性材料で構成されることが好ましい。非生分解性材料として具体的には、PTFE、ポリウレタン、ポリプロピレン、ポリエステル、塩化ビニル、ポリカーボネート、アクリル、ステンレス、チタン、シリコーン、MPCから選択される少なくとも1つ以上から成る材料を挙げることができる。生分解性材料としては、具体的にはポリペプチド、ポリ乳酸、ポリグリコール酸、PLGA、ヒアルロン酸、グリコサミノグリカン、プロテオグリカン、コンドロイチン、セルロース、アガロース、カルボキシメチルセルロース、キチン、キトサンから選択される少なくとも1つ以上からなる材料を挙げることができる。上記の中でも、ポリペプチドが特に好ましい。尚、これら高分子材料には細胞接着性を高める工夫がなされていてもよく、具体的な方法としては1.「基材表面に対する細胞接着基質(フィブロネクチン、ビトロネクチン、ラミニン)や細胞接着配列(アミノ酸一文字表記で現わされる、RGD配列、LDV配列、REDV配列、YIGSR配列、PDSGR配列、RYVVLPR配列、LGTIPG配列、RNIAEIIKDI配列、IKVAV配列、LRE配列、DGEA配列、及びHAV配列)ペプチドによるコーティング」、2.「基材表面のアミノ化、カチオン化」、3.「基材表面のプラズマ処理、コロナ放電による親水性処理」といった方法が利用され得る。
本発明で用いる高分子の「1/IOB」値を上記範囲とすることにより、親水性が高く、かつ、吸水性が高くなることから、栄養成分の保持に有効に作用し、結果として、本発明の細胞3次元構造体(モザイク細胞塊)における細胞の安定化・生存しやすさに寄与するものと推定される。
本発明で用いる高分子のGRAVY値を上記範囲とすることにより、親水性が高く、かつ、吸水性が高くなることから、栄養成分の保持に有効に作用し、結果として、本発明の細胞3次元構造体(モザイク細胞塊)における細胞の安定化・生存しやすさに寄与するものと推定される。
本発明で用いる生体親和性を有する高分子材料は、架橋されているものでもよいし、架橋されていないものでもよいが、架橋されているものが好ましい。架橋方法としては、熱架橋、化学架橋、アルデヒド類(例えば、ホルムアルデヒド、グルタルアルデヒドなど)による架橋、縮合剤(カルボジイミド、シアナミドなど)による架橋、酵素架橋、光架橋、UV架橋、疎水性相互作用、水素結合、イオン性相互作用など公知の方法を用いることができるが、グルタルアルデヒドを用いた架橋法、熱架橋法が好ましい。
のモルモット肝臓由来トランスグルタミナーゼ、ヤギ由来トランスグルタミナーゼ、ウサギ由来トランスグルタミナーゼなど、ヒト由来の血液凝固因子(Factor XIIIa、Haematologic Technologies, Inc.社)などが挙げられる。
本発明にかかる遺伝子組み換えゼラチンとは遺伝子組み換え技術により作られたゼラチン類似のアミノ酸配列を有するポリペプチドもしくは蛋白様物質を意味する。本発明で用いることができる遺伝子組み換えゼラチンは、コラーゲンに特徴的なGly-X-Yで示される配列(X及びYはそれぞれ独立にアミノ酸の何れかを示す)の繰り返しを有するものが好ましい(複数個のGly-X-Yはそれぞれ同一でも異なっていてもよい)。好ましくは、細胞接着シグナルを一分子中に2配列以上含まれている。本発明で用いる遺伝子組み換えゼラチンとしては、コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有する遺伝子組み換えゼラチンを用いることができ、例えばEP1014176A2、US6992172、WO2004-85473、WO2008/103041等に記載のものを用いることができるが、これらに限定されるものではない。本発明で用いる遺伝子組み換えゼラチンとして好ましいものは、以下の態様の遺伝子組み換えゼラチンである。
好ましくは、遺伝子組み換えゼラチンはテロペプタイドを有さない。
好ましくは、遺伝子組み換えゼラチンは天然コラーゲンをコードする核酸により調製された実質的に純粋なコラーゲン用材料である。
(1)配列番号1に記載のアミノ酸配列;又は
(2)配列番号1に記載のアミノ酸配列と80%以上(さらに好ましくは90%以上、最も好ましくは95%以上)の相同性を有し、生体親和性を有するアミノ酸配列;
を有する遺伝子組み換えゼラチンである。
本発明では、上記した生体親和性を有する高分子からなるブロック(塊)を使用する。高分子ブロックの製造方法は特に限定されないが、例えば、高分子からなる固形物を粉砕機(ニューパワーミルなど)を用いて粉砕した後に、ふるいでサイズ分けすることにより所望のサイズのブロックを取得することができる。
本発明で用いる細胞は、本発明の細胞構造体の目的に応じて適宜選択することができ、その種類は特に限定されない。また、細胞は、細胞構造体の使用目的に応じて、1種でも、複数種の組合せて用いてもよい。使用する細胞として、好ましくは、動物細胞であり、より好ましくは脊椎動物由来細胞、特に好ましくはヒト由来細胞である。脊椎動物由来細胞(特に、ヒト由来細胞)の種類は、万能細胞、体性幹細胞、前駆細胞、又は成熟細胞の何れでもよい。万能細胞としては、例えば、ES細胞、GS細胞、又はiPS細胞を使用することができる。体性幹細胞としては、例えば、間葉系幹細胞(MSC)、造血幹細胞、羊膜細胞、臍帯血細胞、骨髄由来細胞、心筋幹細胞、脂肪由来幹細胞、又は神経幹細胞を使用することができる。前駆細胞及び成熟細胞としては、例えば、皮膚、真皮、表皮、筋肉、心筋、神経、骨、軟骨、内皮、脳、上皮、心臓、腎臓、肝臓、膵臓、脾臓、口腔内、角膜、骨髄、臍帯血、羊膜、又は毛に由来する細胞を使用することができる。ヒト由来細胞としては、例えば、ES細胞、iPS細胞、MSC、軟骨細胞、骨芽細胞、骨芽前駆細胞、間充織細胞、筋芽細胞、心筋細胞、心筋芽細胞、神経細胞、肝細胞、ベータ細胞、線維芽細胞、角膜内皮細胞、血管内皮細胞、角膜上皮細胞、羊膜細胞、臍帯血細胞、骨髄由来細胞、又は造血幹細胞を使用することができる。また、細胞の由来は、自家細胞又は他家細胞の何れでも構わない。複数種の細胞を組合せて使用する場合には、例えば、血管系の細胞と他の細胞が挙がられる。血管系の細胞とは、血管内皮細胞、血管内皮前駆細胞、造血幹細胞等がある。血管系の細胞と他の細胞とを組合せることにより、血管を本発明の細胞構造体に誘導することができ、栄養、酸素等を供給することができる。
本発明においては、上記した生体親和性を有する高分子ブロックと上記した細胞とを用いて、複数個の細胞間の隙間に複数個の該高分子ブロックをモザイク状に3次元的に配置させることによって、外部から細胞3次元構造体の内部への栄養送達を可能とし、十分な厚みを有した細胞3次元構造体を形成することに成功した。同時に、細胞3次元構造体の外周部に存在する細胞の働きによって、細胞3次元構造体同士の自然融合を可能とした。
本発明の細胞構造体は、生体親和性を有した高分子材料からなる塊(「ブロック」)と、細胞とを交互に配置することにより製造できる。製造方法は特に限定されないが、好ましくは高分子ブロックを形成したのち、細胞を播種する方法である。具体的には、生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートすることによって、本発明の細胞構造体を製造することができる。例えば、容器中、容器に保持される液体中で、細胞と、予め作製した生体親和性を有する高分子ブロックをモザイク状に配置する。配置の手段としては、自然凝集、自然落下、遠心、攪拌を用いることで、細胞と生体親和性基材からなるモザイク状の配列形成を、促進、制御することが好ましい。
(1)別々に調整したモザイク状細胞塊同士を融合させる、又は
(2)分化培地又は増殖培地下でボリュームアップさせる、
などの方法により所望の大きさの細胞構造体を製造することができる。融合の方法、ボリュームアップの方法は特に限定されない。
遺伝子組み換えゼラチン(リコンビナントゼラチン)として以下記載のCBE3を用意した(WO2008-103041に記載)。
CBE3
分子量:51.6kD
構造: GAP[(GXY)63]3G
アミノ酸数:571個
RGD配列:12個
イミノ酸含量:33%
ほぼ100%のアミノ酸がGXYの繰り返し構造である。CBE3のアミノ酸配列には、セリン、スレオニン、アスパラギン、チロシン及びシステインは含まれていない。CBE3はERGD配列を有している。
等電点:9.34、GRAVY値:-0.682、1/IOB値:0.323
GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G
基材ブロックとして、リコンビナントゼラチンCBE3を用いて、不定形のμブロックを作製した。1000mgのリコンビナントゼラチンを9448μLの超純水に溶解し、1N HClを152μL添加後、終濃度1.0%となるように、25%グルタルアルデヒドを400μL添加し、50℃で3時間反応させ、架橋ゼラチンゲルを作製した。この架橋ゼラチンゲルを、1Lの0.2Mグリシン溶液へ浸漬し、40℃2時間振とうさせた。その後、架橋ゼラチンゲルを、5Lの超純水中で1時間振とう洗浄、超純水を新しい物へ置換し、再び洗浄1時間、を繰り返し、計6回洗浄した。洗浄後の架橋ゼラチンゲルを、-80℃で5時間凍結させた後、凍結乾燥機(EYELA、FDU-1000)で凍結乾燥を行った。得られた凍結乾燥体を、ニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm及び53~106μmのリコンビナントゼラチンμブロックを得た。
基材ブロックとして、天然ゼラチン(Nippi、ニッピゼラチン・ハイグレードゼラチンAPAT)を用いて、不定形のμブロックを作製した。1000mgの天然ゼラチンを9448μLの超純水に溶解し、1N HClを152μL添加後、終濃度1.0%となるように、25%グルタルアルデヒドを400μL添加し、50℃で3時間反応させ、架橋ゼラチンゲルを作製した。この架橋ゼラチンゲルを、1Lの0.2Mグリシン溶液へ浸漬し、40℃2時間振とうさせた。その後、架橋ゼラチンゲルを、5Lの超純水中で1時間振とう洗浄、超純水を新しい物へ置換し、再び洗浄1時間、を繰り返し、計6回洗浄した。洗浄後の架橋ゼラチンゲルを、-80℃で5時間凍結させた後、凍結乾燥機(EYELA、FDU-1000)で凍結乾燥を行った。得られた凍結乾燥体を、ニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm及び53~106μmの天然ゼラチンμブロックを得た。
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM-CDTM BulletKitTM)にて50万cells/mLに調整し、実施例2で作製したリコンビナントゼラチンμブロックを1.0mg/mLとなるように加えた後、100μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、18時間静置し、直径1mm程度の球状の、リコンビナントゼラチンμブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.002μgの高分子ブロック)。その後、培地を軟骨分化培地(タカラバイオ:hMSC Differentiation BulletKitTM,Chondrogenic、TGF-β3)(200μL)へ置換した。Day7で、直径(=厚さ)が1.54mmの球状に、モザイク細胞塊が形成された(図1)。なお、本モザイク細胞塊は、U字型のプレート中で作製するため、球状に作製されている。培地交換は、Day3、7、10、14、17、21で行う。
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM-CDTMBulletKitTM)にて50万cells/mLに調整し、実施例3で作製した天然ゼラチンμブロックを1.0mg/mLとなるように加えた後、100μLをスミロンセルタイトX96Uプレートに播種し、18時間静置し、直径1mm程度の球状の、天然ゼラチンμブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.002μgの高分子ブロック)。その後、培地を軟骨分化培地(タカラバイオ:hMSC Differentiation BulletKitTM,Chondrogenic、TGF-β3)(200μL)へ置換した。Day7で、直径(=厚さ)1.34mmの球状に、モザイク細胞塊が形勢された(図2)。尚、本モザイク細胞塊は、U字型のプレート中で作製するため、球状に作製されている。
実施例4で作製したリコンビナントゼラチンμブロックを用いたモザイク細胞塊について、組織切片を作製した。実施例4で作製した培地中のモザイク細胞塊に対して、培地を除去後、200μLのPBSを加え洗浄し、PBSを除去した。この洗浄工程を2回繰り返した後、洗浄したモザイク細胞塊を10%ホルマリンに浸漬し、2日間ホルマリン固定を行った。その後、パラフィンで包埋し、組織切片を作製した。切片はHE染色(ヘマトキシリン・エオシン染色)し、細胞とゼラチンμブロックの状態を解析した。結果を図3、図4及び図5に示す。これにより、ゼラチンμブロックと、細胞がモザイク状に配置された3次元構造体が作製されていること、さらに細胞が正常な状態でモザイク細胞塊中に存在していることが確認できた。また、この断面切片から、少なくとも厚さ720μm以上のモザイク細胞塊が作製できていることが示された。
実施例4で作製したモザイク細胞塊が、融合可能であるか、つまりモザイク細胞塊を並べていくことで、自然融合し、より大きな3次構造体を形成できるかについて実施した。実施例4で作製した6日目のモザイク細胞塊2個、3個、及び4個をスミロンセルタイトX96Uプレート中で並べ、5日間培養を行った。その結果、モザイク細胞塊同士の間を、外周部に配された細胞が結合させることで、モザイク細胞塊が自然に融合することが明らかになった。図6に実体顕微鏡で撮像した写真を示す。融合開始日(Day6と記載)のモザイク細胞塊では、モザイク細胞塊同士が隣り合って配置されているだけであるが、融合開始から5日目(Dya11と記載)には、モザイク細胞塊間に新たな層が形成され、融合されていっている様子がわかる。また、図7,8,9,10,11には、該融合モザイク細胞塊の断面について、組織切片を作製し、HE染色した結果を示す(固定は10%ホルマリン、包埋はパラフィン包埋)。細胞と、細胞により産生された細胞外基質で、モザイク細胞塊間に融合層が形成されており、モザイク細胞塊同士を融合、結合していることがわかる。これにより、本発明にて作製されるモザイク細胞塊は、自然に融合可能であり、融合させることで、より大きな構造体を形成できることが示された。従って、本発明を用いることで、厚さを有した細胞シート状に作製することも、より立体的な3次元構造体を作製することも可能であることが分かる。
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM-CDTM BulletKitTM)にて50万cells/mLに調整し、実施例2で作製したリコンビナントゼラチンμブロックを1.0mg/mLとなるように加えた後、100μLをスミロンセルタイトX96Uプレートに播種し、18時間静置し、直径1mm球状のモザイク細胞塊を作製した(細胞1個当たり0.002μgの高分子ブロック)。その後、培地を200μLへ増やし、3日毎に培地を交換し培養した。Day7で、直径(=厚さ)1.34mmの球状に、モザイク細胞塊が形成された(尚、本モザイク細胞塊は、U字型のプレート中で作製するため、球状に作製される)。Day7のモザイク細胞塊の切片写真を図16、17に示す。この断面切片上で、厚さの薄い箇所でも、少なくとも624μm以上の厚みを達成していることが分かる。
実施例8で作製した3日目(Day3)のモザイク細胞塊について、培地交換の際、実施例2で作製した0.1mgのリコンビナントゼラチンμブロックを、増殖培地(タカラバイオ:MSCGM-CDTM BulletKitTM)に懸濁して添加した。以後、Day7,10,14,17,21の培地交換に合わせ、0.1mgずつのリコンビナントゼラチンμブロックを添加していった。
実施例4で作製した3日目(Day3)のモザイク細胞塊について、培地交換の際、実施例2で作製した0.1mgリコンビナントゼラチンμブロック(0.1mg)を、軟骨分化培地(タカラバイオ:hMSC Differentiation BulletKitTM,Chondrogenic、TGF-β3)に懸濁して添加した。以後、Day7,10,14,17,21の培地交換に合わせ、0.1mgずつのリコンビナントゼラチンμブロックを添加していった。
実施例4及び実施例5で作製したモザイク細胞塊(hMSC細胞+リコンビナントゼラチン、hMSC細胞+天然ゼラチン)、及び細胞のみで作製した細胞塊(実施例4と同様の手法で、ゼラチンブロックを入れずに作製した)、について、モザイク細胞塊中のグリコサミノグリカン量を定量した。測定は、(Farndale et al., Improved quantitation and sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochimica et Biophysica Acta 883 (1986) 173-177)のDimetylmethylene blue dyeを用いる方法で行い、試薬は硫酸化GAG定量キット(生化学バイオビジネス)を用いた。530nmの吸光を測定し、定量した。図21に示すように、該手法によって、特徴的な吸収ピークが525-530nmに見られることを確認している。
各モザイク細胞塊中の細胞が産生・保持しているATP(アデノシン三リン酸)量を定量した。ATPは生物全般のエネルギー源として知られ、ATP合成量・保持量を定量することで、細胞の代謝活性の状態、活動状態を知ることができる。測定には、CellTiter-Glo(Promega社)を用いた。比較は、実施例4及び実施例5で作製したモザイク細胞塊(hMSC細胞+リコンビナントゼラチン、hMSC細胞+天然ゼラチン)、及び細胞のみで作製した細胞塊(実施例4と同様の手法で、ゼラチンブロックを入れずに作製した)、について、ともにDay7のもので、CellTiter-Gloを用いて、各モザイク細胞塊中のATP量を定量した。
PLGA(乳酸・グリコール酸共重合体: Wako、PLGA7520)0.3gをジクロロメタン(3mL)に溶解した。概PLGA溶解液を乾燥機(EYELA、FDU-1000)にて真空乾燥し、PLGAの乾燥体を得た。PLGA乾燥体を、ニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で10秒×20回の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm及び53~106μmのPLGAμブロックを得た。
PLGA:「1/IOB」値:0.0552
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM-CDTM BulletKitTM)にて50万cells/mLに調整し、実施例13で作製したPLGAμブロックを加えた(最終濃度で0.1mg/mL、0.2mg/mL、1.0mg/mL、2.0mg/mLとなるように条件を振って作成した)後、100μLをスミロンセルタイトX96Uプレートに播種し、18時間静置し、直径1mm弱・球状のモザイク細胞塊を作製した(細胞1個当たり0.0002、0.0004、0.002、0.004μgの高分子ブロック)。その後、培地を200μLへ増やし、3日毎に培地を交換し培養した。尚、本モザイク細胞塊は、U字型のプレート中で作製するため、球状に作製される。Day2のPLGAモザイク細胞塊の実体顕微鏡写真を図24に示す。
アガロース粉末5gを超純水(100mL)に加え、電子レンジを用いて加熱し溶解した。得られた5%アガロース溶解液を常温に戻すことで固形物にして、-80℃で5時間凍結させた後、凍結乾燥機(EYELA、FDU-1000)で凍結乾燥を行うことで、アガロースの凍結乾燥体を得た。アガロース凍結乾燥体を、ニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で10秒×20回の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm及び53~106μmのアガロースμブロックを得た。
IOB値:3.18
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM-CDTM BulletKitTM)にて50万cells/mLに調整し、実施例15で作製したアガロースμブロックを加えた(最終濃度で0.1mg/mL、1.0mg/mLとなるように条件を振って作成した)後、100μLをスミロンセルタイトX96Uプレートに播種し、18時間静置し、直径1mm弱・球状のモザイク細胞塊を作製した(細胞1個当たり0.0002、0.002μgの高分子ブロック)。その後、培地を200μLへ増やし、3日毎に培地を交換し培養した。尚、本モザイク細胞塊は、U字型のプレート中で作製するため、球状に作製された。
新生児SDラット心筋細胞(rCMC)を心筋細胞用培地(Primary Cell Co., Ltd:CMCM 心筋細胞用培養メディウム)にて50万cells/mLに調整し、実施例2で作製したリコンビナントゼラチンμブロックを0.5、1.0、3.0mg/mLとなるように加えた後、それぞれ100μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、18時間静置し、直径1~2mm程度のリコンビナントゼラチンμブロックとrCMC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001、0.002、0.006μgの高分子ブロック)。培地交換は、Day3、7、10、14、17、21で行った。
GFPを発現しているヒト臍帯静脈内皮細胞(GFP-HUVEC:Angio-Proteomie社)を内皮細胞用培地(クラボウ:Medium 200S、LSGS、抗菌剤GA溶液)にて50万cells/mL調整し、実施例2で作製したリコンビナントゼラチンμブロックを0.3、1.0、3.0mg/mLとなるように加えた後、それぞれ100μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種した(細胞1個当たり0.0006、0.002、0.006μgの高分子ブロック)。また、同様にして、細胞を150万cells/mLに調整し、実施例2で作製したリコンビナントゼラチンμブロックを1.0mg/mLとなるように加えた後、100μL、200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種したものも作製した。全てについて、18時間静置し、直径1~2mm程度のリコンビナントゼラチンμブロックとGFP-HUVEC細胞からなるモザイク細胞塊を作製した。培地交換は、Day3、7、10、14、17、21で行った。
Claims (33)
- 生体親和性を有する高分子ブロックと細胞とを含み、該複数個の細胞間の隙間に複数個の該高分子ブロックが配置されている、細胞構造体。
- 該高分子ブロックの大きさが1μm以上700μm以下である、請求項1に記載の細胞構造体。
- 該高分子ブロックの大きさが10μm以上300μm以下である請求項2に記載の細胞構造体。
- 厚さ又は直径が400μm以上3cm以下である、請求項1~3のいずれか一項に記載の細胞構造体。
- 厚さ又は直径が720μm以上1cm以下である、請求項4に記載の細胞構造体。
- 前記高分子ブロックと前記細胞との比率が、細胞1個当り0.0000001μg以上1.0μg以下である請求項1~5のいずれか一項に記載の細胞構造体。
- 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートすることによって製造される、請求項1から6の何れか1項に記載の細胞構造体。
- 生体親和性を有する高分子が、生分解性材料である、請求項1から7の何れか1項に記載の細胞構造体。
- 生体親和性を有する高分子が、ポリペプチド、ポリ乳酸、ポリグリコール酸、PLGA、ヒアルロン酸、グリコサミノグリカン、プロテオグリカン、コンドロイチン、セルロース、アガロース、カルボキシメチルセルロース、キチン、又はキトサンである、請求項1から8の何れか1項に記載の細胞構造体。
- 生体親和性を有する高分子が、ゼラチン、コラーゲン、エラスチン、フィブロネクチン、プロネクチン、ラミニン、テネイシン、フィブリン、フィブロイン、エンタクチン、トロンボスポンジン、又はレトロネクチンである、請求項1から9の何れか1項に記載の細胞構造体。
- 生体親和性を有する高分子が架橋されている、請求項1から10の何れか1項に記載の細胞構造体。
- 架橋がアルデヒド類、縮合剤、又は酵素により施される、請求項11に記載の細胞構造体。
- 生体親和性を有する高分子が、遺伝子組み換えゼラチンである、請求項1から12の何れか1項に記載の細胞構造体。
- 前記生体親和性を有する高分子が、細胞接着性シグナルを一分子中に2以上有する請求項13に記載の細胞構造体。
- 遺伝子組み換えゼラチンが、
式:A-[(Gly-X-Y)n]m-B
(式中、Aは任意のアミノ酸又はアミノ酸配列を示し、Bは任意のアミノ酸又はアミノ酸配列を示し、n個のXはそれぞれ独立にアミノ酸の何れかを示し、n個のYはそれぞれ独立にアミノ酸の何れかを示し、nは3~100の整数を示し、mは2~10の整数を示す。なお、n個のGly-X-Yはそれぞれ同一でも異なっていてもよい。)で示される、請求項13に記載の細胞構造体。 - 遺伝子組み換えゼラチンが、
式:Gly-Ala-Pro-[(Gly-X-Y)63]3-Gly
(式中、63個のXはそれぞれ独立にアミノ酸の何れかを示し、63個のYはそれぞれ独立にアミノ酸の何れかを示す。なお、63個のGly-X-Yはそれぞれ同一でも異なっていてもよい。)で示される、請求項13又は15に記載の細胞構造体。 - 遺伝子組み換えゼラチンが、(1)配列番号1に記載のアミノ酸配列、又は(2)配列番号1に記載のアミノ酸配列と80%以上の相同性を有し、生体親和性を有するアミノ酸配列を有する、請求項13から16の何れか1項に記載の細胞構造体。
- 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程を含む、請求項1から17の何れかに記載の細胞構造体の製造方法。
- 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程において培地交換を行う、請求項18に記載の方法。
- 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程において、培地を分化培地又は増殖培地に交換する、請求項19に記載の方法。
- 生体親和性を有する高分子ブロックと細胞含有培養液との混合物をインキュベートする工程において、生体親和性を有する高分子ブロックをさらに添加する、請求項18から20の何れか1項に記載の方法。
- 請求項18から21の何れか1項に記載の方法により製造される、細胞構造体。
- 請求項1から17の何れかに記載の細胞構造体の複数個を融合することによって得られる、細胞構造体。
- 請求項1から17の何れかに記載の細胞構造体の複数個を融合する工程を含む、請求項23に記載の細胞構造体の製造方法。
- 生体親和性を有する複数個の高分子ブロックと、複数個の細胞とを含み、該複数の細胞により形成される複数個の隙間の一部または全部に、一または複数個の前記高分子ブロックが配置されている細胞構造体を複数個融合させる工程を含む、細胞構造体の製造方法。
- 前記高分子ブロックの大きさが1μm以上700μm以下である、請求項25に記載の細胞構造体の製造方法。
- 前記融合前の各細胞構造体の厚さ又は直径が10μm以上1cm以下であり、前記融合後の厚さ又は直径が400μm以上3cm以下である、請求項25または26記載の細胞構造体の製造方法。
- 生体親和性を有する複数個の第一の高分子ブロックと、複数個の細胞とを含み、該複数の細胞により形成される複数個の隙間の一部または全部に、一または複数個の前記高分子ブロックが配置されている細胞構造体に、更に、第二の高分子ブロックを添加しインキュベートする工程を含む、細胞構造体の製造方法。
- 前記第一の高分子ブロックの大きさが1μm以上700μm以下である、請求項28に記載の細胞構造体の製造方法。
- 前記第二の高分子ブロックの大きさが1μm以上700μm以下である、請求項28または29に記載の細胞構造体の製造方法。
- 前記第二の高分子ブロックを添加しインキュベートした後の、厚さ又は直径が400μm以上3cm以下である、請求項28から30のいずれか一項に記載の細胞構造体の製造方法。
- 請求項25~31のいずれか一項に記載の細胞構造体の製造方法により製造される、細胞構造体。
- 細胞移植、細胞培養または毒性評価用途に使用する、請求項32記載の細胞構造体。
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JPWO2011108517A1 (ja) | 2013-06-27 |
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US20120329157A1 (en) | 2012-12-27 |
KR101744040B1 (ko) | 2017-06-07 |
CN102858381B (zh) | 2015-09-30 |
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EP2543397B1 (en) | 2019-05-08 |
KR20170005168A (ko) | 2017-01-11 |
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