US20170112880A1 - Cardiac cell culture material - Google Patents

Cardiac cell culture material Download PDF

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US20170112880A1
US20170112880A1 US15/401,832 US201715401832A US2017112880A1 US 20170112880 A1 US20170112880 A1 US 20170112880A1 US 201715401832 A US201715401832 A US 201715401832A US 2017112880 A1 US2017112880 A1 US 2017112880A1
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cardiac
fibroblasts
cells
cell
cardiomyocytes
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Takahiro IWAMIYA
Katsuhisa Matsuura
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Metcela Inc
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Metcela Inc
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Publication of US20170112880A1 publication Critical patent/US20170112880A1/en
Priority to US16/106,723 priority Critical patent/US11801267B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3826—Muscle cells, e.g. smooth muscle cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/58—Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1329—Cardiomyocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Definitions

  • the present invention relates to a cardiac cell culture material and a cell culture substrate on which a wall surface and/or a bottom surface of the culture substrate having the wall surface and/or the bottom surface are coated with the cardiac cell culture material.
  • the present invention relates to an artificial organ material obtained by culturing a cardiac cell by using the cardiac cell culture material, and a method for producing the same.
  • a lot of cardiomyocytes were lost and also fibrous tissue replaces that area which leads to cardiac remodeling and heart failure accompanied with excess hemodynamics stress and neurohumoral stimulation.
  • neurohumoral factors such as angiotensin II and endothelin-1 are well known to contribute to promote the cardiac remodeling via blood pressure elevation, cardiomyocyte apoptosis and local inflammation, cardiac fibroblasts have been reported to secrete those factors.
  • Cardiac fibroblasts are also known to play a critical role in heart developments. Interconnected cellular processes in a cardiac fibroblast form a network of collagen, fibroblasts and myocytes. Although cardiomyocyte proliferation is indispensable process of formation of thick ventricular wall and embryonic cardiac fibroblasts have also been reported to promote myocardial mitotic activity through ⁇ -1 integrin signaling. The cardiac fibroblasts dominant causative substance has been unclear. Herein cardiac fibroblasts multifariously act on heart development and pathogenesis and the importance of understanding of mutual interaction and underlying mechanisms between cardiomyocytes and cardiac fibroblasts have been widely recognized. However the uncertain properties of cardiac fibroblasts were the bottle-neck for it and it is required to reveal functional and molecular biological characteristics of cardiac fibroblasts.
  • Heart tissue engineering is promising methods for not only regenerative medicine, but also tissue models.
  • cell sheet-based cardiac tissue using temperature responsive culture dishes have been developed.
  • layering of cardiac cell sheets containing neonatal rats-derived cardiomyocytes, fibroblasts and endothelial cells on the various types of vascular bed enabled to fabricate three-dimensional vascularized viable cardiac tissue (Non patent documents 1 to 3). Since cell sheet-based tissue engineering does not need any scaffold, it requires some amounts of extracellular matrices to construct cell sheets.
  • fibroblasts are indispensable to fabricate cardiac cell sheets when using purified embryonic stem cell-derived cardiomyocytes (Non patent document 4). Since recent reports have suggested that cell-cell interaction between cardiomyocytes and non-myocytes is important for heart physiology and pathogenesis (Non patent document 5), fibroblasts function might also affect the function of the engineered cardiac tissue and it might be prerequisite to select the suitable fibroblasts to fabricate the cardiac tissue in vitro for tissue models. However it remains unclear whether cardiac fibroblasts have the specific function for cardiomyocytes compared with other types of fibroblasts and the related molecular mechanisms.
  • fibroblasts As mentioned above, since the cardiac fibroblasts play an important role in heart developments, and the onset or cure of heart diseases, it is required to separate cardiac fibroblasts that specifically act on cardiac cells such as cardiomyocytes from other fibroblasts, and to sample the cardiac fibroblasts. According to the recent studies, it has been revealed that fibroblasts, which were previously considered as a uniform cell type, have a great variety of phenotypes, and that the phenotypes differ depending on a load state of existing organs, tissues or cells.
  • fibroblasts are only cells morphologically classified. Therefore, among fibroblasts, it is difficult to select only one type thereof having a specific function.
  • VCAM-1 vascular cell adhesion molecule-1
  • CD106 vascular cell adhesion molecule-1
  • ⁇ 4 integrin vascular cell adhesion molecule-1
  • Kwee, et al. reported that VCAM-1 was expressed on embryonic day 11.5 at epicardium, cardiomyocytes, ventricular septum, and the like. It was also reported that, although the expression of ⁇ 4 integrin was recognized at similar areas as those of VCAM-1, ⁇ 4 integrin was not expressed in ventricular septum (Non patent document 6). Moreover, it was reported that, on embryonic day 11.5, there are embryonic death resulting from inhibition of formation of the placenta, and deformity due to decrease in dense layers of ventricular myocardium and ventricular septum in an embryo that is defective in VCAM-1. Yang, et al.
  • Non patent document 7 also reported an epicardium defect in ⁇ 4 integrin null embryo of embryonic day 11.5 (Non patent document 7). Accordingly, it is considered that VCAM-1 and ⁇ 4 integrin mainly contribute to formation of cardiac cells and epicardium in the embryonic stage.
  • One purpose of the present invention is to provide a cardiac cell culture material which specifically acts on cardiac cells, and to provide a cell culture substrate on which a wall surface and/or a bottom surface of the culture substrate having the wall surface and/or the bottom surface are coated with the cardiac cell culture material.
  • another purpose of the present invention is to provide an artificial organ material obtained by culturing a cardiac cell by using the cardiac cell culture material, and a method for producing the same.
  • a functional cardiac tissue is well constructed by using a cardiac cell culture material containing VCAM-1 protein. Therefore, the cardiac cell culture material is coated on a wall surface and/or a bottom surface of a culture substrate having the wall surface and/or the bottom surface, and can be used as a cell culture substrate.
  • a cardiac cell cultured by using the cardiac cell culture material can be used as an artificial organ material.
  • the present invention includes followings.
  • a cardiac cell culture material comprising VCAM-1 protein.
  • VCAM-1 protein is a VCAM-1 separated and purified from an animal material, a VCAM-1 recombinant protein, or a cell expressing VCAM-1 protein.
  • the cardiac culture material according to [1] or [2] used for culturing to construct a cardiac tissue.
  • the cell expressing VCAM-1 protein is a fibroblast expressing VCAM-1 protein.
  • a method of producing an artificial organ material comprising a step of co-culturing a cardiac cell with the cardiac cell culture material according to [1]-[6].
  • a cardiac-derived fibroblast expressing VCAM-1 protein [12] A method of producing the artificial organ material according to [9] further including a step of separating and collecting a cultured cell from a culture substrate. [13] A method of producing the artificial organ material according to [12], wherein the culture substrate is a temperature responsive culture dish, and wherein the separation is performed by temperature change. [14] A method of producing the artificial organ material according to [9] further including the step of agglomerating the co-cultured material by using scaffold having thickness in a certain extent. [15] A method of producing the artificial organ produced from the artificial organ material according to [9] comprising step of using a 3D printer.
  • a functional cardiac cell which can be used in a regenerative medicine and an organizational model can be constructed by culturing a cardiac cell by using the cardiac cell culture material of the present invention.
  • the cardiac cell culture material can be coated on a wall surface and/or a bottom surface of a culture substrate having the wall surface and/or the bottom surface, which can be used as a cell culture substrate. Further, a cardiac cell or a cardiac tissue obtained by culturing can be used as an artificial organ material.
  • FIG. 1 A microscopic observation of NCF, ACF and ADF (photographs).
  • A Bright field microscope images of each fibroblast.
  • B-E Representative Figures of DDR2, vimentin and ⁇ SMA expression (Most of the fibroblasts were not expressing calponin, cytokeratin 11 or NG 2).
  • FIG. 2 Differences in characteristics of mESC derived cardiac cell sheets that were co-cultured with fibroblasts (photographs).
  • A Before separated, many cell masses that were autonomously beating were observed on NCF and ACF co-culture sheet. After decrease in temperature, cell sheet formation was not observed in mESC derived cardiomyocytes and fibroblasts ( ⁇ ).
  • B Extracellular action potentials on each of the cell sheets. Action potentials in ACF or NCF co-culture sheet were observed in each channel. However, the action potentials occurred on a one-off basis on the ADF co-culture sheet (encircling lines indicate the shapes of the cell sheets).
  • C Immunofluorescent stain in each of the cell culture dishes which were observed by a confocal microscope.
  • YFP emitted green (yellow) fluorescence YFP: excitation wavelength 514 nm, fluorescence wavelength 527 nm
  • vimentin emitted red fluorescence cy3: excitation wavelength 512 nm, fluorescence wavelength 552 nm
  • the nucleus was stained in hoechst 33258 (blue)
  • hoechst 33258 excitation wavelength 352 nm, fluorescence wavelength 461 nm.
  • the confocal microscopy observation suggested that, the cells co-cultured with NCF or ACF have a large number of YFP (+) cells, compared with the cells co-cultured with fibroblasts ( ⁇ ) or ADF.
  • the confocal microscopy observation suggested that, the cells co-cultured with NCF or ACF have a large number of cTnT (+) cells, compared with the cells co-cultured with fibroblasts ( ⁇ ) or ADF.
  • E The bar graphs show increase in the numbers of YFP (+) cells or of cTnT (+) cells in each of the cell culture dishes.
  • the numbers of YFP (+) cells or of cTnT (+) cells in fibroblasts ( ⁇ ) were set to 1. More numbers of YFP (+) cells and cTnT (+) cells were observed in NCF or ACF culture dish compared with those in the culture dish of ADF co-culture or fibroblasts ( ⁇ ).
  • there is no significant relationship in the number of cardiomyocytes between NCF and ACF. (N 3, **P ⁇ 0.01)
  • FIG. 3 The number of cardiomyocytes at day 1 and day 5 from the cell culture start in each of the cell culture dishes (Photographs).
  • A Immunofluorescent stain at day 1 from culture start in each of the cell culture dishes which were used in a confocal microscope. YFP emitted green (yellow) fluorescence (YFP: excitation wavelength 514 nm, fluorescence wavelength 527 nm), and cTnT emitted red fluorescence (cy3: excitation wavelength 512 nm, fluorescence wavelength 552 nm), and the nucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence wavelength 461 nm).
  • the bar graphs show increase in the numbers of YFP (+) cells and of cTnT (+) cells (The values at day 1 in fibroblasts ( ⁇ ) were set to 1).
  • more numbers of cardiomyocytes were observed at day 5 from culture start compared with those at day 1.
  • No significant difference was observed between ACF and NCF. (N 3, **P ⁇ 0.01)
  • FIG. 4 Evaluation of proliferation in cardiomyocytes by immunofluorescent stain (photographs).
  • A Immunofluorescent stain observation of Ki67 positive cardiomyocytes in each of co-culture dishes by using the confocal microscope.
  • cTnT was stained by Cy5 (Cy5: excitation wavelength 650 nm, fluorescence wavelength 530 nm), and Ki67 emitted red fluorescence (cy3: excitation wavelength 512 nm, fluorescence wavelength 552 nm), and the nucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence wavelength 461 nm).
  • cTnT was stained by Cy5 (Cy5: excitation wavelength 650 nm, fluorescence wavelength 530 nm), and phosphorylated histone 3 (phosphor S10; Phh3) emitted red fluorescence (cy3: excitation wavelength 512 nm, fluorescence wavelength 552 nm), and the nucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence wavelength 461 nm).
  • YFP emitted green (yellow) fluorescence YFP: excitation wavelength 514 nm, fluorescence wavelength 527 nm
  • cTnT emitted red fluorescence cy3: excitation wavelength 512 nm, fluorescence wavelength 552 nm
  • the nucleus was stained in hoechst 33258 (blue)
  • hoechst 33258 excitation wavelength 352 nm, fluorescence wavelength 461 nm.
  • H The bar graphs show increase in the numbers of YFP (+) cells and of cTnT (+) cells at day 1 and at day 5.
  • the numbers of YFP (+) cells and of cTnT (+) cells at day 1 were set to 1.
  • E Immunofluorescence stain of the VCAM-1 receptor ( ⁇ 4 ⁇ 1) on mESC derived cardiomyocytes.
  • F Western blot analysis of the VCAM-1 receptor on mESC derived cardiomyocytes. The following transient overexpression cell lysate was used as a positive control: Jurkat whole cell lysate.
  • FIG. 6 Identification of cardiac growth factor by immunofluorescence stain analysis (photographs).
  • A-B Immunofluorescence stain observation of the effect of neutralizing antibodies on cardiomyocytes at day 5.
  • YFP emitted green (yellow) fluorescence
  • YFP excitation wavelength 514 nm, fluorescence wavelength 527 nm
  • cTnT emitted red fluorescence
  • cy3 excitation wavelength 512 nm, fluorescence wavelength 552 nm
  • the nucleus was stained in hoechst 33258 (blue) (hoechst 33258: excitation wavelength 352 nm, fluorescence wavelength 461 nm).
  • YFP emitted green (yellow) fluorescence
  • YFP excitation wavelength 514 nm, fluorescence wavelength 527 nm
  • cTnT emitted red fluorescence
  • cy3 excitation wavelength 512 nm, fluorescence wavelength 552 nm
  • cardiomyocyte growth effect was obtained by culturing with VCAM-1 soluble protein (10 ⁇ g/mL).
  • FIG. 7 The results of FACS analysis of cardiac fibroblasts derived from neonatal mice.
  • A-C The results of staining with an anti-VCAM-1 antibody are shown.
  • D The result of a negative control by staining dermal fibroblasts only with a secondary antibody is shown.
  • FIG. 8 The optimum compounding concentration of VCAM-1 (+) cardiac fibroblasts.
  • A Phase difference images of VCFs (VCAM-1 (+)) and VNCFs (VCAM-1 ( ⁇ )) isolated by a magnetic cell separator (MACS).
  • B Fluorescence images (photographs) of VCFs (VCAM-1 (+)) and VNCFs (VCAM-1 ( ⁇ )) isolated by a magnetic cell separator (MACS).
  • VCAM-1 emits red fluorescence
  • FIG. 9 Evaluation of optimum compounding ratio of VCFs.
  • ADFs mouse adult-derived dermal fibroblasts
  • VCFs VCAM-1 positive mouse neonatal cardiac fibroblasts
  • VNCFs VCAM-1 negative mouse neonatal cardiac fibroblasts
  • NCFs neonatal mouse cardiac fibroblasts.
  • GFP cardiomyocytes emit green fluorescence, and Ki 67 emits red fluorescence, and cTnT was stained by Cy5 (Cy5: excitation wavelength 650 nm, fluorescence wavelength 530 nm). Magnification is ⁇ 20.
  • FIG. 11 Evaluation of division (proliferation) effect, migration effect and ability of constructing a network of cardiomyocytes for 5 days of culturing under the condition of co-culturing with NCFs and ADFs (photographs).
  • YFP expressing cardiomyocytes emit green fluorescence
  • DsRed fibroblasts emit red fluorescence. Magnification is ⁇ 200.
  • FIG. 12 A shows fluorescence images indicating division (proliferation) of cardiomyocytes for three days of culturing at the time when co-culturing ES-derived cardiomyocytes with VCFs (VCAM-1 (+)) or VNCFs (VCAM-1 ( ⁇ )) (photographs).
  • VCFs VCAM-1 (+)
  • VNCFs VCAM-1 ( ⁇ )
  • B shows the results evaluating the migratory ability by calculating the total migratory distance for 3 days of culturing at the time when co-culturing ES-derived cardiomyocytes, and VCFs or VNCFs.
  • N 5, *P ⁇ 0.05.
  • FIG. 13 The results of analyzing the localization of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) and VCAM-1 positive cardiac fibroblasts with flow cytometry.
  • the present embodiment relates to a cardiac cell culture material containing VCAM-1.
  • the “cardiac cell culture material” may be any material that is used when culturing a cardiac cell.
  • the material includes but is not limited to a reagent such as a protein, and a peptide, etc. to be added to a culture medium, and a material, etc. for coating a bottom surface or a wall surface of a culture substrate of a culture vessel, etc. such as a petri dish and a flask, and the like.
  • these cell culture substrate, in which a wall surface and/or a bottom surface of the culture substrate having the wall surface and/or the bottom surface are coated with the cardiac cell culture material include microcarrier, and cell culture bag, etc.
  • VCAM-1 vascular cell adhesion molecule-1
  • vascular cell adhesion molecule-1 is a known protein as a cell adhesion molecule that expresses in a vascular endothelial cell, and the like.
  • VCAM-1 includes but not limited to a protein encoded by a gene described in accession number NM_001078, etc. of NCBI (National Center for Biotechnology Information), and also includes an isoform obtained by alternative splicing.
  • the VCAM-1 protein in the present embodiment includes VCAM-1 which is expressing on a cell surface, a soluble VCAM-1, various mutants one or a plurality of, for example, 1-20, 1-15, 1-10 or 1-5 of amino acids of which have been deleted from, substituted from, or added to an amino acid of VCAM-1 protein and having the same activity as VCAM-1 protein.
  • a VCAM-1 protein in an animal material which has been separated and purified by a well-known method and a recombinant protein may be used as the VCAM-1 in the present embodiment.
  • the animal material includes but is not limited to humans; experimental animals such as mice, rats, guinea pigs, hamsters, pigs, monkeys and rabbits; and bacteria such as E. coli , etc.
  • a commercially available recombinant protein may be also used.
  • a cell that is expressing VCAM-1 may be used as VCAM-1 of the present embodiment.
  • a publicly known cell sorting method may be used.
  • the cell sorting method includes but not limited to flow cytometry using an anti-VCAM-1 antibody, magnetic bead method, affinity column method, and panning method.
  • Anti-VCAM-1 antibodies are not particularly limited. Commercially available anti-VCAM-1 antibodies may be used, and a product produced by a known method by using VCAM-1 as an antigen may be also used. Moreover, as far as the cells that are expressing VCAM-1 may be screened, either monoclonal antibody or polyclonal antibody may be used; however, it is preferred to use monoclonal antibody from the viewpoint of specificity.
  • the methods of screening the cardiac cell culture materials of the present embodiment include, a step of preparing cells, a step of performing cell sorting to the cells by using a VCAM-1 antibody, and a step of collecting only cells that have been judged to be expressing VCAM-1 as a result of the cell sorting.
  • the types are not limited as far as VCAM-1 is expressed. However, it is preferred to use fibroblasts.
  • the fibroblasts include all the cells that will ultimately become fibroblasts or myofibroblasts. Namely, the scope of fibroblasts of the present embodiment includes the cells that are in the middle of differentiation or a maturation stage and cannot be identified as fibroblasts or myofibroblasts at that time as far as the cells will ultimately become fibroblasts or myofibroblasts.
  • fibroblasts of the present embodiment includes the cells that are not called as fibroblast, such as stromal cells, interstitial cells, progenitor cells, precursor cells, stem cells, or the like, as far as the cells have the same functions and activities as fibroblasts and express VCAM-1.
  • fibroblasts are not limited. Pluripotent stem cells such as ES cells, iPS cells and muse cells, and adult stem cells such as mesenchymal stem cells may be differentiated and used, and primary cells taken from animals may be used, and established cells may be used. However, cardiac-derived fibroblasts are preferably used, and among them, epicardium-derived fibroblasts are in particular preferred to be used. In a case where established cells are used, processing of cell sorting may be omitted by selecting the cells that are known to express VCAM-1. The animals from which fibroblasts are derived may be appropriately selected in accordance with the animals from which the cells to be co-cultured are derived.
  • the animals include humans; experimental animals such as mice, rats, guinea pigs, hamsters, pigs, monkeys and rabbits; pet animals such as dogs, cats and birds; and livestock such as cattle, horses, sheep and goats.
  • the fibroblasts may be of at any time of the animals such as fetus, neonate, infant, adult, and there is no limit.
  • the cardiac cell culture material of the present embodiment may be a composition containing physiological saline, cell culture solution, or cell preservation solution, etc. for maintenance or preservation of VCAM-1 protein or cells that are expressing VCAM-1 protein.
  • the contents contained in the composition there is no limit on the contents contained in the composition as far as the contents do not impair the function of VCAM-1.
  • the state of the cardiac cell culture material of the present embodiment may be liquid, gel-like, freezed, or freeze-dried, and the state thereof is not limited.
  • the cardiac cell culture material may include fibroblasts regardless of presence or absence of VCAM-1 protein.
  • the fibroblasts include all the cells that will ultimately become fibroblasts or myofibroblasts. Namely, even if the cells are in the middle of differentiation or a maturation stage and cannot be identified as fibroblasts or myofibroblasts at that time, if the cells are those that will ultimately become fibroblasts or myofibroblasts, the cells may be used without limit. Among them, fibroblasts that are expressing CD31 (vascular endothelial cell marker) are preferred.
  • the ratio of VCAM-1 protein expressing cells (cell number):CD31 expressing cells (cell number) are preferably 5:5-9:1, more preferably 5:5-8.2, even more preferably 6:4-8.2, and may be 7:3-8:2.
  • the present embodiment relates to an artificial organ material in which the ratio of cardiac fibroblasts expressing VCAM-1 to all the cells is 50% or higher.
  • the ratio of the cardiac fibroblasts is 50% or higher, and preferably 60% or higher, and more preferably 70% or higher, and even more preferably 80% or higher, and most preferably 90% or higher.
  • the artificial organ material may be obtained by co-culturing cardiac fibroblasts that express VCAM-1 with cardiomyocytes, and the ratio of the cardiac fibroblasts at the start of co-culturing is normally 3% or higher, preferably 4% or higher, more preferably 6% or higher, and even more preferably 8% or higher, and most preferably 9% or higher.
  • the ratio of the cardiac fibroblasts at the start of co-culturing is preferably 30% or lower, preferably 20% or lower, and most preferably 20%.
  • the present invention also relates to an artificial organ material obtained by culturing a cardiac cell with the above-mentioned cardiac cell culture material, or a method for producing the same.
  • the cardiac cell culture material of the present embodiment can construct a functional cardiac tissue that can be used in a regenerative medicine and organizational model by culturing with a cardiac cell.
  • the cardiac tissue can be used as an artificial organ material.
  • the artificial organ material can be of any form. For example, it can be adhered to a damaged part of organs such as heart in the form of sheet.
  • one embodiment is a method for curing heart diseases by attaching the sheet to a damaged part of the heart.
  • the artificial organ material can be also transplanted to a defect site of an organ after it is laminated or it is agglomerated by using scaffold, which has thickness in a certain extent.
  • the material of the scaffold includes but not limited to hydroxyapatite, atelocollagen, and gel.
  • the artificial organ material can be used for cell transplantation, academic research, etc. as it is in the state of the culture cell, without making it to be a particular form.
  • an artificial organ can be produced from the artificial organ material by using a 3D printer. The produced artificial organ not only can be used for transplantation but also can be widely used for safety pharmacology test and preclinical research, etc.
  • constructing a cardiac tissue means constructing a tissue having at least one of the cardiac functions such as promoting division of cardiac cells, and providing uniform beating throughout a whole tissue, which can be used for regenerative medicine and a tissue model.
  • the cardiac functions include all the known cardiac functions such as autonomous pulsating ability, contraction and relaxation ability, impulse conduction ability, and hormone secretion ability, etc.
  • the cardiac functions are not limited to functions which only the heart has. For example, a muscle cell also has the contraction and relaxation ability. However, even if other cells have an equivalent function, it does not affect the definition of the cardiac functions of the present embodiment. Further, with respect to the cardiac functions, there is no limit on highness and lowness of the functions as long as they are suitable for use purpose of a cardiac tissue.
  • contraction and relaxation ability For example, for the purpose of producing an artificial heart, it is required to have a contraction and relaxation ability to the extent that it can pump out blood thought out the body; however, for the purpose of academic research, etc. of contraction and relaxation ability in vitro, it is satisfied if contraction and relaxation ability is detected by some means.
  • cardiac cells to be used include all the cells that constitute the heart such as cardiomyocytes, smooth muscle cells, pacemaker cells and vascular endothelial cells.
  • the derivation of the cardiac cells can be appropriately set in accordance with the purpose of use as an artificial organ material.
  • human-derived cardiac cells may be used, and for the purpose of constructing a tissue model in a mouse experiment, mouse-derived cardiac cells may be used.
  • a cardiac cell of any period from fetus, newborn, pediatric and adult can be used, and there is no limit on the period.
  • the cardiac cell of the present embodiment is preferred to be produced from pluripotent stem cells such as ES cells, iPS cells, and muse cells, and adult stem cells such as mesenchymal stem cells.
  • the “culturing” of the present embodiment can be carried out by a publicly known cell culturing method, and there is no limit on the condition of the culturing as long as a cardiac cell culture material and a cardiac cell are present in a culture vessel, or are immersed in the same culture medium.
  • the cardiac cell culture material are cells which are expressing VCAM-1 protein
  • the mixing percentage of the cells (cell number) that are expressing VCAM-1 to cardiac cells are preferably 3-20%, more preferably 6-18% and most preferably 9-16%.
  • a culture liquid used for the culturing can be appropriately set in accordance with a kind of cell to be cultured.
  • DMEM fetal calf serum
  • ⁇ -MEM fetal calf serum
  • RPMI-1640 fetal calf serum
  • Nutritional substances such as FCS and FBS and antibiotics may be added to the culture liquid.
  • Growth factor and cytokines such as fibroblast growth factors (FGF) may also be added to the culture liquid.
  • FGF fibroblast growth factors
  • the number of days until the desired cell number and/or function are obtained may be appropriately set.
  • the periods include 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months.
  • the cultivation temperature may be appropriately set in accordance with the kinds of cells to be cultured.
  • the temperature may be 10-60° C., preferably 20-50° C., and more preferably 30-40° C.
  • the production method of the present embodiment may further include the step of collecting cultured cells.
  • the “cultured cells” may include both fibroblasts and cardiac cells, and may only include the cardiac cells.
  • the cell may be separated and collected by using proteases such as trypsin.
  • proteases such as trypsin.
  • Wild-type C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan).
  • B6 Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Tokyo Women's Medical University.
  • the following antibodies were used for immune cytochemistry, western blot and flow cytometric analysis (FACS): rabbit polyclonal anti-discoidindomein receptor tyrosine kinase 2 (DDR2) (GeneTex, Irvine, Calif.); guinea pig monoclonal anti-vimentin (Progen, Heidelberg, Germany); mouse monoclonal anti-NG2 (Millipore, Temecula, Calif.); Rabbit polyclonal anti-alpha smooth muscle actin (Abcam, Cambridge, UK); mouse monoclonal anti-cardiac troponin T (cTnT) (Thermo Scientific, Rockford, Ill.); mouse monoclonal anti-cytokeratin11 (EXBIO, NadSafinou, CZ); rabbit polyclonal anti-Ki67 (Abcam, Cambridge, UK); rabbit polyclonal anti-Histon H3 (phosphor S10) (Abcam, Cambridge, UK); rat monoclonal anti-integrin ⁇ 4/ ⁇ 1
  • mESC expressing the neomycin phosphotransferase gene under the control of the ⁇ -myosin heavy chain promoter and cardiomyocyte differentiation and purification were described previous report (Matsuura K, et al. Biomaterials. 2011; 32:7355-7362). Briefly, for cardiac induction and cardiomyocyte purification, trypsinized ES cells were seeded at 5 ⁇ 10 4 cells/mL (total, 125 mL/flask) into spinner flasks (Integra Biosciences, Zizers, Switzerland) and cultured with DMEM supplemented with 10% FBS for 10 days, then these differentiated cells were treated with neomycin for further 8 days.
  • Fibroblasts were obtained from Wild-type C57BL/6 mice (Neonatal, 1 day; Adult, 10-12 weeks).
  • Neonatal cardiac fibroblasts were obtained from hearts of neonatal mice (1 day of age) as described previous report (Matsuura K, et al, Biomaterials. 2011; 32: 7355-7362). NCFs from passage 3 were used for the experiments.
  • ACFs Adult cardiac fibroblasts
  • mice 10-12 weeks
  • First hearts were washed with PBS ( ⁇ ) and cut into circa 5 mm 2 species. These species were covered with sterilized cover glasses and cultured with DMEM supplemented with 10% FBS on 10 cm culture dishes. 2 weeks after starting culture, cells were dissociated with 0.25% Trypsin/EDTA and subcultured to other 10 cm dishes. ACFs from passage 3 were used for the experiments.
  • ADFs Adult dermal fibroblasts
  • mice were obtained from dorsal dermal tissue of adult mice (10-12 weeks).
  • First harvested dermal tissues were treated with Dispase I [1000 U/mL] (Eidea inc.) over night at 4° C.
  • the tissues were cut into circa 1 mm 2 species. These species were covered with sterilized cover glasses and cultured with DMEM supplemented with 10% FBS on 10 cm culture dishes. 2 weeks after starting culture, cells were dissociated with 0.25% Trypsin/EDTA and subcultured to another 10 cm dishes.
  • ADFs from passage 3 were used for the experiments.
  • NCFs and ADFs were isolated from B6.Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice (Neonatal: 1 day, Adult: 10 weeks) with the same methods as described above.
  • mESC-derived cardiomyocytes were co-cultured with each type of fibroblasts at the ratio of 8:2 with DMEM supplemented with 10% FBS (3.2 ⁇ 10 5 cells/cm 2 ). After 5 days of culture, the cells were incubated at 20° C. for detaching cell sheets. Bright field images of samples were obtained by a Nikon ECLIPSE Ti (Nikon, Tokyo, Japan).
  • the electrical activities of the cardiomyocyte sheets were obtained from the extracellular potentials measured by a multi-electrode array (MED) system (Alpha MED Sciences inc.) as described previous report (Matsuura K, et al, Biomaterials. 2011; 32:7355-7362).
  • MED multi-electrode array
  • BrdU staining for a FACS analysis was performed as described in a BrdU Flow Kits Instruction Manual (BD Pharmingen, Franklin Lakes, N.J.). Briefly, cells were fixed and permeabilized with BD Cytofix/Cytoperm Buffer, then exposed incorporated BrdU with DNase. BrdU staining was performed with APC-anti-BrdU antibody (BD Pharmingen, Franklin Lakes, N.J.). Samples were analysed with a Gallios (Beckman Coulter, Brea, Calif.).
  • BD Cytofix/Cytoperm Buffer (BD Pharmingen, Franklin Lakes, N.J.); BD Perm/Wash Buffer (10 ⁇ ) (BD Pharmingen, Franklin Lakes, N.J.); BD Cytoperm Plus Buffer (10 ⁇ ) (BD Pharmingen, Franklin Lakes, N.J.); BrdU (10 mg/mL) (BD Pharmingen, Franklin Lakes, N.J.); DNase (BD Pharmingen, Franklin Lakes, N.J.).
  • RNA quantity and quality were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Mass.) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, Calif.), as recommended.
  • cDNA products were then used as templates for in vitro transcription to generate fluorescent cRNA.
  • cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40° C. for 2 hours.
  • Labeled cRNAs were purified using QIAGEN's RNeasy mini spin columns and eluted in 30 ⁇ l of nuclease-free water. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a Nanodrop ND-1000 spectrophotometer and an Agilent Bioanalyzer.
  • the altered transcripts were quantified using the comparative method. We applied 2-fold change in signal intensity to identify the significant differences of gene expression in this study.
  • RT-PCR included 10 minutes at 25° C., 120 minutes at 37° C., and 5 seconds at 85° C. with iCycler (BIO-RAD).
  • cDNA template (1 ⁇ g) was used from each sample.
  • TaqMan probe real-time PCR studies were performed with TaqMan Gene Expression Assays (Applied biosystems). All experiments were conducted in triplicate. Samples were cycled 40 times with an 7300 Real Time PCR System (Applied Biosystems) as follows: 2 minutes at 50° C. and 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Relative quantification was calculated according to the ⁇ CT method for quantitative real-time PCR using a Gap DH gene as endogenous control.
  • NCFs or ADFs were lysed in Laemmli sample buffer (BIO-RAD, CA, USA), protease inhibitor (Boehringer Mannheim, Indianapolis, Ind.) and 2-mercaptoethanol (Wako Pure Chemical Industries, Japan).
  • the samples were separated on a 4% to 12% Bis-Tris Gels (Life Technologies, MD, United States), electrotransferred to a iBlot Transfer Stack, nitrocellulose, regular-size (Life technologies, MD, United States) with iBlot 7-Minute Blotting System (Life technologies, MD, United States), and processed for chemiluminescence analysis with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, PA, United States).
  • fibroblasts were seeded onto the upper layer of insert culture dishes (2.4 ⁇ 10 5 cells).
  • mESC-derived cardiomyocytes were seeded onto the below layer (4.8 ⁇ 10 5 cells).
  • the culture medium with the antibody at 10 ⁇ g/mL was changed every day until 5 days.
  • FIGS. 3A to C the number of cardiomyocytes was examined at day 1 and day 5 in co-culture.
  • the number of cardiomyocytes was identical among conditions, suggesting that each type of fibroblasts did not affect the initial adherence of cardiomyocytes after seeding.
  • the number of YFP (+) and cTnT (+) cardiomyocytes at day 5 was significantly higher than that at day 1.
  • the number of cardiomyocytes at day 5 was similar to that at day 1.
  • integrin ⁇ 4 ⁇ 1 is known to be the principal co-receptor of VCAM-1
  • VCAM-1 VCAM-1 recombinant protein
  • VCAM-1 positive cells To confirm importance of VCAM-1 positive cells in constructing functional cardiac cell sheets, we measured the percentage of VCAM-1 positive cells in organism-derived cardiac fibroblasts.
  • Cardiac fibroblasts were dissected and collected from neonatal mice (1 day) of C57/BL6 mice, and skin fibroblasts were dissected and collected from adult mice (10-12 weeks). Each of the fibroblasts were adhesion-cultured up to passage 3, and the cell volume of 1 ⁇ 10 7 cells per condition was obtained. Passage 3 is the same condition with the culture condition of the above-mentioned cardiomyocytes produced by cell sheets.
  • FIGS. 7A-C The results of cardiac fibroblasts (NCFs) were shown in FIGS. 7A-C . It was found that the percentage of VCAM-1 positive cells in NCFs was approximately 60% ( FIG. 7A : 66.57%, FIG. 7B : 58.95%, FIG. 7C : 54.73%). Conversely, the percentage of VCAM-1 positive cells in skin fibroblasts (ADFs) was approximately 5%, and it turned out that the percentage of VCAM-1 positive cells in NCFs is significantly more than that of ADFs (P ⁇ 0.001).
  • VCAM-1 positive cardiac fibroblasts originate from an outer membrane-derived cell from the view point of embryology, and we obtained the suggestion that it is effective to classify fibroblasts from the view point of embryology, and not to conduct morphological classification but to conduct functional classification as a cell source for constructing a functional tissue.
  • NCFs vascular endothelial cell marker
  • EMT epithelial mesenchymal transition
  • EndMT endothelial mesenchymal transition
  • kidney fibroblasts that differentiate from vascular endothelial cells through EndMT are expressing CD31 (J Am Soc Nephrol 19:2282-2287, 2008). This may also become one of the bases for supporting that NCFs are expressing CD31.
  • cardiac fibroblasts enhance proliferation of mouse embryonic stem cell (mESC) derived cardiomyocytes, and contribute to construction of more functional cardiac cell sheets. Moreover, it was indicated that cardiac fibroblasts are more abundantly expressing VCAM-1 compared with skin fibroblasts, and that the VCAM-1 of cardiac fibroblasts play an important role in proliferation of cardiac cells and construction of cardiac tissues that are functionally biologically-designed.
  • mESC mouse embryonic stem cell
  • Example 1 reveals that when a myocardial tissue derived from a pluripotent stem cell is constructed, cardiac fibroblasts cause cell proliferation of cardiomyocytes through VCAM-1 which is protein highly expressed by the cardiac fibroblasts, prompt beating in the created whole myocardial tissue, and significantly improve the functionality. However, it is also revealed that cardiac fibroblasts have a heterozygous character even in a local area named heart, and that all the cardiac fibroblasts do not necessarily express VCAM-1.
  • VCAM-1 VCAM-1
  • Wild-type C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan).
  • B6 Cg-Tg (CAG-DsRed*MST) 1Nagy/J mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). All the experimental protocols were approved by The Keio University Institutional Animal Care and Use Committee. The following antibodies were used for immunofluorescent staining and flow cytometry.
  • mice monoclonal anti-cardiac troponin T (cTnT) (Thermo Scientific, Rockford, Ill.),
  • mESC mouse ES cells
  • YFP yellow fluorescent protein
  • Mouse ES-derived cardiomyocytes (Cor.At) to which puromycin resistance gene and green fluorescent protein (GFP) were introduced under ⁇ -myosin heavy chain promoter were purchased from Axiogenesis AG (Cologne, Germany).
  • Mouse ES-derived cardiomyocytes were treated with puromycin for 2 days, and were cultured in a medium not containing puromycin for 2 weeks.
  • VCFs Cardiac fibroblasts isolated from a one day old wild-type C57BL/6 neonatal mouse were cultured, and VCFs were isolated with a magnetic cell sorter (Magnetic-activated cell sorting, MACS). The isolated VCFs were re-cultured, and an experiment was carried out after removing dead cells.
  • MACS Magnetic-activated cell sorting
  • Tissues of the heart collected from a wild-type C57BL/6 mouse were dissociated with gentleMACS Octo Dissociator (Miltenyi Biotec, Gladbach, Germany), and homogenized to the cellular level.
  • the obtained cells were immunofluorescent-stained, and subsequently analyzed with an S3 cell sorter (BIO-RAD, CA).
  • YFP positive ES cell-derived cardiomyocytes and cardiac fibroblasts isolated from a DsRed mouse were co-cultured, and the inside of a BZ-9000 fluorescent microscope was kept at the concentration of 5% CO2 at 37° C., and time-lapse observation was carried out for 5 days (Keyence, Osaka, Japan).
  • GFP expressing type cardiomyocytes were seeded at the concentration of 5.45 ⁇ 10 4 cells/cm 2 ′ and fibroblasts isolated with MACS were seeded at the concentration of 1.36 ⁇ 10 4 cells/cm 2 , which were weaker than the concentration at the time when a myocardial tissue was constructed, and time-lapse photography was carried out with a BZ-X700 fluorescent microscope for 3 days. The photographed time-lapse images were analyzed with a Motion Analyzer so as to calculate the total migratory distance (mm) of cardiomyocytes for three days of culturing, and the evaluation of the migratory ability was carried out (Keyence).
  • VCFs VCAM-1 negative cardiac fibroblasts
  • VNCFs VCAM-1 negative cardiac fibroblasts
  • VCFs and VNCFs collected from cardiac fibroblasts and dermal fibroblasts were co-cultured with cardiomyocytes at each compounding ratio (Table 1), the highest mitogenic effect of cardiomyocytes was observed at Day 5 under the condition where 20% of VCFs and 80% of cardiomyocytes were seeded ( FIGS. 9A and 9B ).
  • FIGS. 9, 11 and 12 It was revealed that, when a myocardial tissue was constructed by co-culturing at the concentration of 20% VCFs and 80% cardiomyocytes, cardiomyocytes grew at the highest level in the myocardial tissue, and obtained high migratory ability ( FIGS. 9, 11 and 12 ). Further, it was revealed that 9.5% cardiomyocytes and 90.5% fibroblasts existed in a myocardial tissue created under the above-mentioned condition ( FIG. 10 ). In order to evaluate whether the localization of cardiomyocytes and fibroblasts was different from a biological heart, a heart of a one day old mouse was collected, and crushed into the cellular level by enzyme treatment, and the evaluation of localization of cardiomyocytes and VCFs were carried out with flow cytometry.
  • VCFs existed at 14.8% in the heart, and that 55.6% of fibroblasts expressed VCAM-1 protein ( FIGS. 13, 14A and 14B ). Moreover, it was revealed that fibroblasts that express CD31 existed at 3.7% in the heart, and that 16.1% of cardiac fibroblasts expressed CD31 protein ( FIGS. 13, 14C and 14D ).
  • the present study revealed that the optimum seeding concentration of VCFs required to create a high functional myocardial tissue was 20% by evaluating the proliferation level of cardiomyocytes by compounding VCFs sorted by a magnetic cell separator (Magnetic-activated cell sorting, MACS) and ES cell-derived cardiomyocytes at each concentration.
  • MACS Magnetic-activated cell sorting
  • VCFs provide cardiomyocytes with high migratory ability, and it was suggested that the provision of high migratory ability takes part in formation of a high level myocardial network in a myocardial tissue.
  • cardiac cells obtained by the culture can be used as regenerative medicines such as transplantation, or as artificial organ materials such as cardiac tissue models.

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