US20160177270A1 - Method for integrating biological tissues with a vascular system - Google Patents

Method for integrating biological tissues with a vascular system Download PDF

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US20160177270A1
US20160177270A1 US14/906,699 US201414906699A US2016177270A1 US 20160177270 A1 US20160177270 A1 US 20160177270A1 US 201414906699 A US201414906699 A US 201414906699A US 2016177270 A1 US2016177270 A1 US 2016177270A1
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cells
tissue
tissues
vascular
mouse
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Takanori Takebe
Hideki Taniguchi
Yoshinobu Takahashi
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Yokohama City University
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • C07D499/21—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring with a nitrogen atom directly attached in position 6 and a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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Definitions

  • the present invention relates to a method of biological tissues with a vascular system. More specifically, the present invention relates to a method of preparing three-dimensional tissues with vascular networks from tissues induced from pluripotent stem cells, etc. or tissues (such as normal or cancer tissue) isolated from individuals.
  • Non-Patent Document No. 1 Takayama K, et al., Biomaterials. 2013 February; 34(7):1781-9;
  • Non-Patent Document No. 2 Saito H, et al., PLoS ONE. 2011; 6(12): e28209;
  • Non-Patent Document No. 3 Eiraku M, et al., Nature 2011, 472, 51-56).
  • none of the tissues induced by those methods have vasculatures.
  • Vasculatures have such a role that, once transplanted, they supply the tissues with oxygen and nutrients that are necessary for their survival. What is more, it is believed that, even before blood flows into the tissue, recapitulating three-dimensional tissue structures with blood vessels and cell polarity as well is important for the differentiation, proliferation and maintenance of cells. Therefore, avascular tissues not only fail to engraft upon transplantation and suffer from inner necrosis, but also fail to achieve tissue maturation that is associated with vascularization. It has, therefore, been difficult for avascular tissues to exhibit adequate functions.
  • Non-Patent Document No. 4 Kaufman-Francis K, et al., PLoS ONE 2012, 7(7): e40741).
  • the present inventors have established an innovative three-dimensional culture technique which realized “directed differentiation of organ cells based on organ reconstitution”, by utilizing spatiotemporal interactions between different cell lineages (Nature, 499:481-484, 2013; WO2013/047639 titled “Method for Producing Tissue and Organ”). Briefly, by recapitulating those intracellular interactions among organ cells, vascular cells and mesenchymal cells which are essential for early processes of organogenesis, a primordium of steric organ (an organ bud) is induced, thus providing a platform for enabling the generation of functional organs with vascular networks.
  • this method starts with organ cells and it has not been elucidated as to whether a primordium of three-dimensional tissue with vascular networks can be generated by using a tissue fragment(tissue).
  • the present invention aims at providing a method of constituting a tissue construct with vasculatures in vitro from a tissue without depending on scaffold materials.
  • the present inventors have found that close intercellular reactions between organ cells (from which organs develop) and vascular endothelial cells/mesenchymal cells direct the progress of steric tissue formation that involves autonomous tissue structure constitution and cell differentiation (Nature, 499:481-484, 2013; WO2013/047639 titled “Method for Producing Tissue and Organ”). However, it is yet to be made clear if vascular networks can be integrated into tissue fragments.
  • the present invention attempts to artificially generate steric tissues having vascular networks in vitro starting with tissues by artificially recapitulating such early processes of organogenesis. Further, by transplanting the steric tissues into living bodies, the present invention intends to create a vascularized steric tissue which, when transplanted into a living body after being induced in a culture system, restarts blood flow to enable the tissue function to achieve maturation and maintenance.
  • the present inventors have cocultured tissues isolated from individuals (up to approximately 10-3,000 ⁇ m) or tissues induced from pluripotent stem cells (up to approximately 10-3,000 ⁇ m) with vascular cells and mesenchymal cells at appropriate mixing ratios. The methods described below were used for inducing steric tissues.
  • Three-dimensional tissues are formed by coculturing tissues with vascular/mesenchymal cells on a carrier such as Matrigel. 2. Three-dimensional tissues are formed by coculturing tissues with vascular/mesenchymal cells on a plate of such a shape that cells gather in the bottom.
  • the present inventors successfully created tissues/organs with a highly ordered tissue structure comparable to that of adult tissues; when the steric tissues induced in a culture system were by transplanted into living bodies, reconstruction of functional vascular networks was induced, whereupon blood perfusion was restarted to create the above-described tissues/organs.
  • normal/cancer tissues isolated from individuals or tissues induced from pluripotent stem cells are cocultured with vascular cells and mesenchymal cells under appropriate environments, whereby it has become possible to constitute steric tissue constructs in vitro that are integrated with vascular networks. Since vascular networks which are essential for maturation, maintenance, repair, etc. of tissues are provided, highly functional tissues are reconstituted, potentially providing a platform for preparing tissue constructs useful for drug discovery screening and regenerative medicine.
  • tissue constructs obtained from pluripotent stem cells by directed differentiation remained less mature in the differentiation stage than functional cells that constitute adult tissues. This is because terminal differentiation of functional cells has not been achieved by the conventional directed differentiation method.
  • the present invention it has become possible to reconstitute a tissue integrated with vascular networks and one may expect that a method of directing terminal differentiation of human functional cells will be established (for example, reconstitution of cell polarity in vasculature); hence, the present invention is highly valuable as a technique for creating human functional cells.
  • the present invention it is possible to reconstitute a steric human tissue construct having a vascular system. Therefore, it will become possible to generate a tissue or an organ that permits a blood flow in an appropriately arranged vascular system and which has been entirely unachievable by conventional techniques. Consequently, one may expect that the present invention will provide a completely novel analysis system for evaluating the efficacy of pharmaceuticals by which the relationship between development of drug efficacy and blood vessels and other factors that have been difficult to analyze by existing evaluation systems can be evaluated.
  • tissue which are constituted from difficult-to-expand cells (such as pancreatic ⁇ cells, renal glomerular epithelial/renal tubular epithelial cells, hepatic cells, intestinal epithelial cells, alveolar epithelial cells, tumor cells, trophectodermal cells, iPS cell-derived endodermal cells, iPS cell-derived mesodermal cells, iPS cell-derived from ectodermal cells and iPS cell-derived tissue stem/progenitor cells) and examples of such tissues include pancreatic islets, renal glomeruli, liver tissues, intestinal crypts, pulmonary alveoli, tumor tissues, trophectodermal tissues, iPS cell-derived endodermal cell-derived spheroids, iPS cell-derived mesodemial cell-derived spheroids, iPS cells-derived ectodermal cell-derived spheroids and iPS cell-derived tissue stem/
  • difficult-to-expand cells such as pan
  • Tissues can be generated by the method disclosed in Nature, 499:481-484, 2013; WO2013/047639 only in the case where isolated cells are used.
  • the method of the present invention has been confirmed to be capable of integrating a vascular system for tissues, rather than cells, that are approximately 10-3,000 ⁇ m in size. 3.
  • stem cells such as iPS cells
  • normal or cancer tissues isolated from individuals or tissues induced from pluripotent stem cells or the like are cocultured with vascular cells and mesenchymal cells, whereby it has become possible to constitute steric tissue constructs in vitro that are integrated with vascular networks.
  • This technique is applicable to, for example, generation of human functional cells; organ transplantation; drug discovery screening; novel analysis systems to evaluate the relationship between development of drug efficacy and blood vessels.
  • FIG. 1A This figure shows the integration of vascular networks to pancreatic islet (hereinafter, frequently referred to simply as “islet”) tissues.
  • FIG. 1B This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1C This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1DE This figure shows the integration of vascular networks to islet tissues.
  • E Mouse islets at 24 hours of culture.
  • E Mouse islets, vascular endothelial cells and mesenchymal stem cells at 24 hours of coculture.
  • E′ Immunohistological analysis of the three-dimensional tissue generated in E) (green: insulin, red: human CD31).
  • FIG. 1F This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1G This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1H This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1I This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1J-1 This figure shows the integration of vascular networks to islet tissues.
  • J-1) Group of genes whose expressions are markedly enhanced by coculture with vascular endothelial cells and mesenchymal stem cells.
  • FIG. 1J-2 This figure shows the integration of vascular networks to islet tissues.
  • FIG. 1J-3 This figure shows the integration of vascular networks to islet tissues.
  • FIG. 2A This figure shows preparation of vascularized islet fragments.
  • FIG. 2B This figure shows preparation of vascularized islet fragments.
  • FIG. 2C This figure shows preparation of vascularized islet fragments.
  • FIG. 2D This figure shows preparation of vascularized islet fragments.
  • FIG. 2E This figure shows preparation of vascularized islet fragments.
  • mice Histological analysis of vascularized islet fragments; mouse islets (red), vascular endothelial cells (green), mesenchymal stem cells (colorless) and mouse CD31 (blue).
  • FIG. 3AB This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3CD This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3E This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3F This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3G This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3H This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3I This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3JK This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3L This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 3M This figure shows validation of function upon transplantation of vascularized tissue.
  • FIG. 4 This figure shows the integration of vascular networks to renal glomeruli.
  • D Macroscopic image of vascularized three-dimensional mouse renal glomerular tissue at 24 hours of culture using a 96-well dish.
  • E Confirmation of vascularization and engraftment at the site of transplantation of vascularized renal glomeruli.
  • F Live imaging of the site of transplantation of vascularized renal glomeruli (mouse renal glomeruli (red), human vascular endothelial cells (green), mouse vascular endothelial cells (blue)).
  • FIG. 5 This figure shows the integration of vascular networks to tumor tissues.
  • CD44 cancer stem cell marker
  • FIG. 6 This figure shows the integration of vascular networks to liver tissues.
  • FIG. 7 This figure shows the integration of vascular networks to intestinal tissues.
  • FIG. 8 This figure shows the integration of vascular networks to pulmonary tissues.
  • FIG. 9 This figure shows the integration of vascular networks to iPS cell-derived endodermal tissues.
  • the present invention provides a method of integrating a vascular system for a biological tissue in vitro, comprising coculturing a biological tissue with vascular cells and mesenchymal cells.
  • biological tissue refers to a construct constituted from a plurality of cells.
  • normal/abnormal tissues or cancer tissues isolated from individuals as well as tissues induced from pluripotent stem cells (such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells)), tissue stem/progenitor cells, differentiated cells or the like may be enumerated.
  • tissue stem/progenitor cells differentiated cells or the like may be enumerated.
  • biological tissues those derived from humans may primarily be used.
  • Biological tissues derived from non-human animals may also be used.
  • animals used for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like
  • mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like may also be used.
  • vascular system refers to a structure composed of vascular endothelial cells and its supporting cells.
  • Vascular systems not only mainain tissues but also play an important role in the maturation process of tissues.
  • Vascular structures have such a role that, once transplanted, they supply the tissues with oxygen and nutrients that are necessary for their survival. What is more, it is believed that even before blood flows into the tissue, recapitulating three-dimensional tissue structures with blood vessels and cell polarity as well is important for the differentiation, proliferation and maintenance of cells. Therefore, avascular tissues not only fail to engraft upon transplantation and suffer from inner necrosis, but also fail to achieve tissue maturation that is associated with vascularization. It has, therefore, been difficult for avascular tissues to exhibit adequate functions.
  • integrating a vasculature system and “vascularization” mean that a vascular system composed of vascular endothelial cells and its supporting cells is integrated directly with a target tissue.
  • Vascular cells may be isolated from vascular tissues but they are in no way limited to those isolated therefrom.
  • Vascular cells may be derived from totipotent or pluripotent cells (such as iPS cells and ES cells) by induction of differentiation.
  • vascular cells vascular endothelial cells are preferable.
  • the term “vascular endothelial cells” means cells constituting vascular endothelium or cells capable of differentiating into such cells (for example, vascular endothelial progenitor cells and vascular endothelial stem cells).
  • Whether a cell is vascular endothelial cell or not can be determined by checking to see if they express marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3 and CD31 (if any one or more of the above-listed marker proteins are expressed, the cell can safely be regarded as a vascular endothelial cell). Further, as markers for vascular endothelial progenitor cells, c-kit, Sca-1, etc. have been reported. If these markers are expressed, the cell of interest can be confirmed as a vascular endothelial progenitor cell (S. Fang, et al., PLOS Biology, 2012; 10(10): e1001407).
  • marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3 and CD31 (if any one or more of the above-listed marker proteins are expressed, the cell can safely be regarded as a vascular endothelial cell). Further, as markers for vascular endothelial progenitor cells,
  • vascular endothelial cell of the present invention: endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, hemangioblast (H J. Joo, et al. Blood. 25; 118(8):2094-104 (2011)) and so on.
  • vascular cells human-derived cells are mainly used.
  • vascular cells derived from non-human animals may also be used.
  • non-human animals e.g., animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like
  • vascular cells may be obtained from cord blood, umbilical cord vessels, neonatal tissues, liver, aorta, brain, bone marrow, adipose tissues, and so forth.
  • the term “mesenchymal cells” means connective tissue cells that are mainly located in mesodemi-derived connective tissues and which form support structures for cells that function in tissues.
  • the “mesenchymal cell” is a concept that encompasses those cells which are destined to, but are yet to, differentiate into mesenchymal cells.
  • Mesenchymal cells to be used in the present invention may be either differentiated or undifferentiated. Preferably, undifferentiated mesenchymal cells are used.
  • Whether a cell is an undifferentiated mesenchymal cell or not may be confirmed by checking to see if the cell expresses marker proteins such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271 or Nestin (if any one or more of the above-listed marker proteins are expressed, the cell can safely be regarded as an undifferentiated mesenchymal cell).
  • a mesenchymal cell in which none of the above-listed markers is expressed can be judged as differentiated mesenchymal cell.
  • mesenchymal stem cells mesenchymal progenitor cells
  • mesenchymal cells R.
  • mesenchymal cells human-derived cells are mainly used.
  • mesenchymal cells derived from non-human animals e.g., animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like
  • non-human animals e.g., animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like
  • non-human animals e.g., animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the
  • the size of a biological tissue to be cocultured with vascular cells and mesenchymal cells may be approximately 10-500 ⁇ m, but is not limited to this range. Preferably, the size is approximately 100-300 ⁇ m. More preferably, the size is approximately 100-150 ⁇ m.
  • the numbers of vascular cells and mesenchymal cells to be used for coculture may each be about 2 ⁇ 10 2 -1 ⁇ 10 5 cells, preferably, about 2 ⁇ 10 2 -5 ⁇ 10 4 cells, and more preferably, about 1 ⁇ 10 4 cells, per biological tissue of approx. 150 ⁇ m in size.
  • the culture ratio of vascular cells and mesenchymal cells in coculture is not particularly limited if it is within such a range that a vascular system is provided for biological tissues.
  • a preferable cell count ratio as expressed by the vascular cell to mesenchymal cell is 10-3:3-1.
  • the number of biological tissues in coculture is not particularly limited if it is within such a range that a vascular system is provided for biological tissues.
  • a vascular system is provided for biological tissues.
  • 1-100 tissues approx. 100-150 ⁇ m in diameter are used for a mixture of 1 ⁇ 10 4 vascular cells and 1 ⁇ 10 4 mesenchymal cells.
  • Either one or both of vascular cells and mesenchymal cells may be substituted by substances such as factors secreted by vascular cells, factors secreted by mesenchymal cells, and factors secreted as a result of the presence of both vascular cells and mesenchymal cells.
  • factors secreted by vascular cells examples include, but are not limited to, FGF2, FGF5, BMF4, BMP6, CTGF, angiopoietin 2, chemokine (C-C motif) ligand 14 and von Willebrand factor.
  • FGF2 may be added at 10-100 ng/ml, preferably at about 20 ng/ml, per 1 ⁇ 10 6 cells; and BMF4 may be added at 10-100 ng/ml, preferably at about 20 ng/ml, per 1 ⁇ 10 6 cells.
  • the medium used for culturing is not particularly limited. Any medium may be used as long as it enables the integration of a vascular system for biological tissues.
  • a medium for culturing vascular cells in particular, vascular endothelial cells
  • a medium for culturing biological tissues or a mixture of these two media may be used.
  • any medium may be used but, preferably, a medium containing at least one of the following substances may be used: hEGF (recombinant human epithelial growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS, antibiotics (e.g., gentamycin or amphotericin B), heparin, L-glutamine, phenol red and BBE.
  • EGM-2 BulletKit (Lonza), EGM BulletKit (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), human microvascular endothelial cell growth medium (Toyobo) or the like may be used.
  • the medium used for culturing biological tissues is not particularly limited but, as a medium for culturing islet tissues, RPMI1640 (Wako) or EGMTM BulletKitTM (Lonza CC-4133) supplemented with 10% fetal bovine serum (BWT Lot.S-1560), 20 mmol/L L-glutamine (Gibco) and 100 ⁇ g/ml penicillin/streptomycin (Gibco) may preferably be used; as a medium for culturing renal tissues (such as renal glomeruli), RPMI1640 (Wako) supplemented with 20% fetal bovine serum (BWT Lot.S-1560), 100 ⁇ g/ml penicillin/streptomycin (Gibco) and Insulin-Transferrin-SeleniumX (Gibco) may preferably be used; as a medium for culturing intestinal tissues (such as crypt fragments), RPMI1640 (Wako) supplemented with 20%
  • biological tissues are seeded on a substrate such as gel and cocultured with vascular cells and mesenchymal cells.
  • the substrate may be a base material having a stiffness of 0.5-25 kPa.
  • base material include, but are not limited to, gels (e.g., ranging from a stock solution to a 4-fold dilution of MatrigelTM, agarose gel, acrylamide gel, hydrogel, collagen gel or urethane gel).
  • biological tissues may be cocultured with vascular cells and mesenchymal cells on a plate of such a shape that cells gather in the bottom.
  • the plate used for this purpose is not particularly limited as long as it has such a shape that cells gather in the bottom.
  • PrimeSurfaceTM 96-well U plate (Sumitomo Bakelite) may be used.
  • the temperature at the time of culture is not particularly limited but it is preferably 30-40° C., more preferably 37° C.
  • the time period of culture is not particularly limited but it is preferably 12-144 hours.
  • the culture period is more preferably about 12-24 hours.
  • the culture period is more preferably about 48-72 hours.
  • the culture period is more preferably about 12-72 hours.
  • the biological tissue that has been integrated with a vascular system by the method of the present invention may be a construct characterized in that the complex tissue is autonomously formed by cells or tissues. Further, the biological tissue that has been integrated with a vascular system by the method of the present invention may be a complex tissue in which the vascular system directly integrates with (i.e., adheres to, connects to, or continues to) the tissue.
  • vascular system for a biological tissue by coculturing the biological tissue with vascular cells and mesenchymal cells without using scaffold materials.
  • vascular system for a biological tissue by coculturing the biological tissue with vascular cells and mesenchymal cells
  • the function of the biological tissue can be maintained and/or improved.
  • transplantation efficiency is sufficiently improved to provide a treatment method having remarkable therapeutic effects.
  • the present invention which enables reconstruction of a vascular system will leads to the establishment of a method by which terminally differentiated cells can be efficiently induced from tissues derived from pluripotent stem cells such as iPS cells and ES cells.
  • the biological tissue that has been integrated with a vascular system by the method of the present invention may be a complex tissue whose vascular system is capable of rapidly functioning in vivo.
  • a living body host
  • the time it takes for anastomosis to host vessels to occur and for blood to flow in can be greatly shortened, compared to cases where scaffold materials are used [for example, when scaffold materials are used, 12 days are taken (Engineered blood vessel networks connect to host vasculature via wrapping-and-tapping anastomosis. Blood. 2011 Oct. 27; 118(17):4740-9) whereas the method of the present invention takes only 1 to 2 days (see Examples described later)].
  • the present invention provides a method of preparing a tissue or an organ, comprising transplanting a human or a non-human animal with a biological tissue that has been integrated with a vascular system by coculturing with vascular cells and mesenchymal cells, and differentiating the biological tissue into a tissue or an organ in which vascular networks have been constructed.
  • Non-human animals to be used in this method include, but are not limited to, animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like may be used. Further, in order to avoid immunorejection, the non-human animal to be used herein is preferably an immunodeficient animal.
  • the site of transplantation of the biological tissue integrated with a vascular system may be any site as long as transplantation is possible.
  • Specific examples of the transplantation site include, but are not limited to, the intracranial space, the mesentery, the liver, the spleen, the kidney, the subcapsular space of the kidney, and the supraportal space.
  • the biological tissue is to be transplanted into the intracranial space, about 1 to 12 biological tissues of 500 ⁇ m in size, prepared in vitro, may be transplanted.
  • the biological tissue is to be transplanted into the mesentery, about 1 to 12 biological tissues of 3-8 mm in size, prepared in vitro, may be transplanted.
  • biological tissue When the biological tissue is to be transplanted into the supraportal space, about 1 to 12 biological tissues of 3-8 mm in size, prepared in vitro, may be transplanted. When the biological tissue is to be transplanted into the subcapsular space of the kidney, about 1 to 6 biological tissues of 3-8 mm in size, prepared in vitro, may be transplanted. When the biological tissue is to be transplanted into the liver, spleen, kidney, lymph node or blood vessel, about 100-2000 biological tissues of 100-200 ⁇ m in size, prepared in vitro, may be transplanted.
  • the tissues and organs prepared as described above may be used in drug discovery screening and regenerative medicine.
  • the present invention provides a method of regeneration or function recovery or a tissue or an organ, comprising transplanting a human or a non-human animal with a biological tissue that has been integrated with a vascular system by coculturing with vascular cells and mesenchymal cells into, and differentiating the biological tissue into a tissue or an organ in which vascular networks have been constructed.
  • Non-human animals to be used in this method include, but are not limited to, animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like may be used.
  • the present invention provides a composition for regenerative medicine, comprising a biological tissue that has been integrated with a vascular system by coculturing with vascular cells and mesenchymal cells.
  • the composition of the present invention can be transplanted into a living body to prepare a tissue or an organ.
  • the composition of the present invention can also be transplanted into a living body to regenerate a tissue or an organ or recover its function.
  • the living body not only humans but also animals (such as ones used as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, ratfish, salmon, shrimp, crab or the like) may be used.
  • the biological tissue is capable of differentiating into a tissue or an organ having vascular networks.
  • blood perfusion can occur. It is believed that the occurrence of blood perfusion in the vascular networks enables generation of a tissue or an organ having a highly ordered tissue structure either comparable or nearly comparable to the tissue structure of adult tissues.
  • composition of the present invention may contain additives including, for example, tissue vascularization promoters such as FGF2, HGF and VEGF; gelatin sponge for hemostasis associated with transplantation (product name: Spongel; Astellas Pharma); and tissue adhesives used to fix transplanted tissues, such as Bolheal (Teijin Pharma), BeriplastTM (CSL Behring) and TachoCombTM (CSL Behring).
  • tissue vascularization promoters such as FGF2, HGF and VEGF
  • gelatin sponge for hemostasis associated with transplantation product name: Spongel; Astellas Pharma
  • tissue adhesives used to fix transplanted tissues such as Bolheal (Teijin Pharma), BeriplastTM (CSL Behring) and TachoCombTM (CSL Behring).
  • the present invention also provides a method of preparing a non-human chimeric animal, comprising transplanting a non-human animal with a biological tissue that has been integrated with a vascular system by coculturing with vascular cells and mesenchymal cells, and differentiating the biological tissue into a tissue or an organ in which vascular networks have been constructed.
  • the non-human animal (such as mouse) transplanted with the biological tissue integrated with a vascular system can mimic the physiological function of the animal species (such as human) from which the vascularized biological tissue is derived.
  • Non-human animals include, but are not limited to, animals used, for example, as experimental animals, pet animals, working animals, race horses or fighting dogs; more specifically, mouse, rat, rabbit, pig, dog, monkey, cattle, horse, sheep, chicken, shark, devilfish, radish, salmon, shrimp, crab or the like may be used. Further, in order to avoid immunorejection, the non-human animal to be used herein is preferably an immunodeficient animal.
  • the present invention also provides a method of evaluating a drug, comprising using at least one member selected from the group consisting of the biological tissue integrated with a vascular system by the above-described method, the tissue or organ prepared from the vascularized biological tissue, and the non-human chimeric animal transplanted with the vascularized biological tissue.
  • drug evaluation include, but are not limited to, evaluation of drug metabolism (e.g., prediction of drug metabolism profiles), evaluation of drug efficacy (e.g., screening for drugs that are effective as pharmaceuticals; confirmation of the effect of pharmaceuticals such as the relationship between drug efficiency and blood vessels; etc.), toxicity evaluation, and evaluation of drug interactions.
  • human-type drug metabolism profiles may be obtained as follows. Briefly, a biological human tissue integrated with a vascular system, a human tissue or organ prepared from a biological tissue integrated with a vascular system, or a non-human chimeric animal transplanted with a biological human tissue integrated with a vascular tissue is administered with a candidate compound for pharmaceuticals; then, biological samples are taken and analyzed. According to these processes, prediction of the distribution/metabolism/excretion process of pharmaceuticals in humans—which has been extremely difficult to achieve by conventional methods—becomes possible and one may. expect that the development of safe and efficacious pharmaceuticals can be remarkably accelerated.
  • Screening for drugs that are effective as pharmaceuticals is carried out as follows. Briefly, starting with a tissue induced from a cell/tissue established from a diseased patient, a biological tissue integrated with a vascular system, a tissue or an organ prepared from this vascularized biological tissue, or a non-human chimeric animal transplanted with this vascularized biological tissue is prepared. Then, a candidate compound for pharmaceuticals is administered for analyses. As a result, one may expect that the prediction accuracy of drug efficacy in actual administration to humans—which has been insufficient in conventional in vitro tests—can be greatly improved.
  • a biological tissue integrated with a vascular system a tissue or an organ prepared from this vascularized biological tissue, or a non-human chimeric animal transplanted with this vascularized biological tissue is administered with a given drug. Then, the concentration distribution of the drug in tissues at the vicinity of blood vessels and the desired drug's effect on cells are measured.
  • tumor tissues for example, targeting cancer stem cells which are clinically considered a cause of recurrence or metastasis is believed to be an important therapeutic strategy.
  • cancer stem cells which are clinically considered a cause of recurrence or metastasis is believed to be an important therapeutic strategy.
  • vascular permeability is decreased and anticancer agents are difficult to infiltrate whereas if they are distant from blood vessels, diffusion of anticancer agents is insufficient.
  • drugs targeting at cancer stem cells it has been important to reconstitute a three-dimensional tumor tissue that starts from blood vessels and use this tissue for evaluation.
  • the evaluation of drug efficacy based on cell/tissue polarity with respect to blood vessels which has been entirely unachievable by conventional methods can be realized and development of drugs with higher therapeutic effects can be performed.
  • a biological tissue integrated with a vascular system a tissue or an organ prepared from this vascularized biological tissue or a non-human chimeric animal transplanted with this vascularized biological tissue is used as a target which is administered a test substance and thereafter the expressions of tissue disorder markers are measured, whereby the accuracy in disorder prediction can be improved.
  • Evaluation of drug interactions may be performed as follows. Briefly, a biological tissue integrated with a vascular system, a tissue or an organ prepared from this vascularized biological tissue or a non-human chimeric animal transplanted with this vascularized biological tissue is used as a target which is administered with a plurality of drugs; then, examination of each drug's pharmacokinetics (distribution/metabolism/excretion processes), toxicity evaluation, and drug efficacy evaluation are performed.
  • the function level of the cells obtained from pluripotent stem cells by conventional directed differentiation remained less mature in the differentiation stage than those functional cells that constitute adult tissues. If, by the method of the present invention, terminally differentiated functional cells are obtainable from tissues induced from pluripotent stem cells or the like, it will be a revolutionary technique of directed differentiation that serves as an important platform adapted for industrial production of human functional cells. For example, human hepatocytes or human hepatic stem cells isolated from the human liver tissues artificially prepared by the present invention will enable mass production of human adult hepatocytes which are necessary for drug discovery and development.
  • tissue transplantation therapy involving the transplantation of islet tissues or the like extracted from bodies derived from brain-dead donors, for example.
  • engraftment of transplants after the transplantation was remarkably low because the transplants used in tissue transplantation therapy had no vascular system.
  • the therapeutic effect was rather limited.
  • it has become possible to supply vascularized transplants that can solve this problem. If industrial production of human tissues/organs for therapeutic purposes that are integrated with vascular networks becomes possible, new tissues/organs for transplantation which are expected to provide higher therapeutic effects can be supplied, potentially serving as a revolutionary medical technique.
  • mice Isolation of mouse pancreatic islets (hereinafter, frequently referred to simply as “islets”) was performed mainly according to the method of Dong et al. (Title of the document: A protocol for islet isolation from mouse pancreas).
  • C57BL/6J mice Japan SLC, Inc.
  • Wako diethyl ether
  • the ampulla of Vater was ligated.
  • a 27 G injection needle was inserted into the site of junction of the cystic duct and the hepatic duct, and 3 ml of collagenase XI solution (1,000 U/ml) (Sigma, cet. No. C7657) prepared with Hanks' buffer (HBSS, Gibco) was injected to fill the entire pancreas with collagenase XI solution.
  • the pancreas was cut out and placed in a 50 ml tube containing collagenase XI solution, which was then shaken at 37.5° C. for 15 min. After digestion of the pancreas, 25 ml of ice-cooled HBSS (containing 1 mM CaCl 2 ) was added to the tube for washing.
  • the tube was centrifuged (290 g, 30 sec, 4° C.), followed by removal of the supernatant. After re-washing and re-centrifugation, 15 ml of HBSS was added to the tube. The resultant content was filtered with a 70 ⁇ m mesh cell strainer. The residue was entirely transferred into a petri dish using an originally prepared medium [EGMTM BulletKitTM (Lonza CC-4133) originally modified for the purpose of culturing islets].
  • mice islets isolated in 1 above were observed under a stereomicroscope, orange-colored spherical mouse islets (150-250 ⁇ m in diameter) could be confirmed. These islets were transferred to an islet culture medium with a Pipetman.
  • Mouse islets were cultured using an originally prepared medium [EGMTM BulletKitTM (Lonza CC-4133) supplemented with 10% fetal bovine serum (BWT Lot. S-1560), 20 mmol/L L-glutamine (Gibco) and 100 ⁇ g/ml penicillin/streptomycin (Gibco)] in a 37° C. 5% CO 2 incubator.
  • HUVECs normal human umbilical vein endothelial cells
  • HUVECs Human umbilical vein endothelial cells
  • hMSCs Human mesenchymal stem cells
  • MSCGMTM BulletKitTM Lonza PT3001
  • Both HUVECs and hMSCs were cultured in a 37° C., 5% CO 2 incubator.
  • Production of virus vectors pGCD ⁇ NsamEGFP and pGCD ⁇ NsamKO was performed by the method described below. Briefly, 293GPG/pGCD ⁇ NsamEGFP cells (kindly provided by Mr. Masafumi Onodera) and 293GPG/pGCD ⁇ NsamKO cells (kindly provided by Mr. Masafumi Onodera) were seeded on poly-L-lysine-coated dishes and cultured in an especially prepared medium (designated “293GPG medium”).
  • DMEM fetal bovine serum
  • Gibco 2 mmol/L L-glutamine
  • Gabco 1 ⁇ penicillin/streptomycin
  • G418 1 ⁇ g/mL tetracycline hydrochloride
  • T-7660 2 ⁇ g/mL puromycin
  • G418 0.3 mg/mL G418
  • the medium was exchanged with a different medium equivalent to 293GPG medium except that it was freed of tetracycline hydrochloride, puromycin and G418 (this medium is designated “293GP medium”) (the day of exchange shall be day 0).
  • 293GP medium the medium equivalent to 293GPG medium except that it was freed of tetracycline hydrochloride, puromycin and G418
  • the viruses were recovered together with the medium starting at day 4, followed by filling with 293GP medium again.
  • the recovered medium was passed through a 0.45 ⁇ m filter and stored temporarily at 4° C.
  • the medium recovered up to day 7 by the above-described procedures was centrifuged (6000 G, 4° C., 16 hr). To the resultant pellet, 400 ⁇ L of Stempro (Invitrogen) was added. After shaking at 4° C. for 72 hr, the resultant solution was recovered and stored at ⁇ 80° C. (designated “100-fold concentrated virus solution”).
  • HUVECs were cultured until they reached 30-50% confluence.
  • Protamine Sigma
  • pGCD ⁇ NsamEGFP was added to the medium to give a final concentration of 0.4 ⁇ m/mL
  • pGCD ⁇ NsamEGFP was added to HUVECs.
  • cells were infected in a 37° C., 5% CO 2 incubator for 4 hr and washed with PBS twice.
  • the medium was exchanged with a fresh one, followed by incubation in a 37° C., 5% CO 2 incubator again. These operations were repeated four times and the cells were fluorescence labeled.
  • EGFP-HUVECs 2.0 ⁇ 10 6 cells
  • hMSCs 4.0 ⁇ 10 5 cells
  • cells were suspended in 20 ⁇ l of a medium for islets, and gel was solidified [Briefly, Matrigel (BD) and the medium for islets were mixed at 1:1; the resultant solution was poured into each well (300 ⁇ l/well); and the plate was left standing in a 37° C., 5% CO 2 incubator for 10 min or more until solidification occurred].
  • Cells were seeded on each well of a 24-well flat bottom plate (BD) in which 300 mouse islets/well had been left standing. After seeding, the plate was left standing in a 37° C. incubator for 10 min. After 10 minutes, 1 ml of the medium for islets was added gently down the well wall, followed by incubation in a 37° C. incubator for one day.
  • Mouse islets were left standing in each well of PrimeSurfaceTM 96-Well U Plate (Sumitomo Bakelite) preliminarily filled with the medium for islets, and HUVECs and hMSCs were seeded in each well. The plate was subsequently incubated in a 37° C. incubator for one day.
  • PrimeSurfaceTM 96-Well U Plate Suditomo Bakelite
  • Coculture was performed for tracking chronological changes with a stereomicroscope. Briefly, 10 mouse islets were left standing in each well of PrimeSurfaceTM 96-Well U Plate. In each well, HUVECs (1.0 ⁇ 10 4 cells) and hMSCs (1.0 ⁇ 10 3 cells) were seeded. After seeding, the plate was mounted in a stereomicroscope (Leica DFC300FX) to observe morphological changes caused by coculture.
  • a stereomicroscope Leica DFC300FX
  • HUVECs (1 ⁇ 10 5 cells), hMSCs (2 ⁇ 10 4 cells) and a mixture of HUVECs (1 ⁇ 10 5 cells) and hMSCs (2 ⁇ 10 4 cells) were individually seeded in those inserts, which were then placed in the 24-well plates where mouse islets had been left standing.
  • the plates were incubated in a 37° C., 5% CO 2 incubator overnight.
  • 200 ⁇ l of LIVE/DEADTM Cell Imaging Kit (Life Technologies, Japan) was added to each well of the 24-well plates where mouse islets had been left standing. Then, the plates were incubated in a 37° C., 5% CO 2 incubator for 15 min, followed by observation under a confocal microscope (LEICA TCS-SP5).
  • Mouse islets (100) were left standing in the bottom of each well of 24-well Transwell plates. Inserts were placed in other 24-well plates. Inserts in which a mixture of HUVEC (1 ⁇ 10 5 cells) and hMSC (2 ⁇ 10 4 cells) was seeded and inserts in which no cell was seeded were prepared. These inserts were placed in the 24-well plates where mouse islets had been left standing. Then, the plates were incubated in a 37° C., 5% CO 2 incubator overnight. Subsequently, supernatant was collected from the 24-well plates where mouse islets had been left standing, and subjected to measurement with an insulin measurement kit (Shibayagi; Cat. No. AKRIN-011H).
  • an insulin measurement kit Shibayagi; Cat. No. AKRIN-011H
  • Glucose-free RPMI1640 (Wako) was prepared as a medium for islets. By adding glucose, a low glucose medium (60 mg/100 ml) and a high glucose medium (360 mg/100 ml) were created. The low glucose medium was filled in the inserts of 24-well Transwell plate where mouse islets (100) had been left standing. The inserts were transferred to wells where a mixture of HUVECs (1 ⁇ 10 5 cells) and hMSCs (2 ⁇ 10 4 cells) had been seeded, followed by incubation in a 37° C., 5% CO 2 incubator for 1 hr.
  • the medium in the inserts was exchanged with the high glucose medium, and the inserts were transferred to other wells, followed by incubation in an incubator for 1 hr. After incubation, supernatants from inserts and wells were collected and subjected to measurement with an insulin measurement kit (Shibayagi).
  • NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used as transplantation animal were bred under a SPF environment with a light-dark cycle consisting of 10 hours for day and 14 hours for night.
  • the breeding of experimental animals were entrusted to the Animal Experiment Center, Joint Research Support Section, Advanced Medical Research Center, Yokohama City University. Animal experiments were performed in accordance with the ethical guidelines stipulated by Yokohama City University.
  • CW mice Preparation of CW mice was performed mainly according to the method of Yuan et al. (Document Title: Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows).
  • ketalar Sudyo Yell Yakuhin Co., Tokyo, Japan
  • xylazine Sigma Chemical Co., St. Louis, Mo., USA
  • Ketalar was used according to the Narcotics Administration Law.
  • mice After confirmation of the absence of bleeding, the surface of the brain was filled with physiological saline (Otsuka Pharmaceutical Co., Tokyo, Japan). Then, a custom-made circular slide glass 7 mm in diameter (Matsunami, Osaka, Japan) was mounted on the surface and sealed tightly with an adhesive prepared by mixing coatley plastic powder (Yoshida, Tokyo, Japan) with Aron Alpha (Toagosei Co., Tokyo, Japan) until the mixture became cementitious. One week after the preparation of CW, those mice which did not have any sign of bleeding or inflammation at the site of surgery were selected and used in the subsequent experiments.
  • Diabetes model mice were created by administering diphtheria toxin (DT) to SCID Ins-TRECK-Tg mice (kindly provided by Tokyo Metropolitan Institute for Clinical Medicine). DT 1 ⁇ g/kg was adjusted with physiological saline to give a dose of 200 ⁇ l per mouse and injected intraperitoneally. After administration, regular glucose level and body weight were measured every day at 17:00. Those mice which had a regular glucose level reading of 300 mg/dl for consecutive three days or more were used as diabetes model mice. Measurement of glucose levels was performed by Glutest neo SensorTM (Panasonic, Tokyo) on blood samples taken from the tail vein.
  • Glutest neo SensorTM Panasonic, Tokyo
  • mice prepared in Section 15 above underwent transplantation after their brain surfaces were exposed by removing the glass of the cranial window. Those mice which did not have any sign of bleeding, inflammation or infection on their brain surfaces were used. After anesthetization, the area surrounding the cranial window was disinfected with 70% ethanol. The pointed end of an 18G needle was inserted into the border line between the custom-made circular slide glass and Aron Alpha and so manipulated as to peel off the slide glass without damaging the brain surface. Thus, the brain surface was exposed. Subsequently, the brain surface was washed with physiological saline. A tissue transplant was left standing near the center of the brain surface, and the slide glass was remounted.
  • the space between the slide glass and the brain surface was filled with physiological saline and thereafter the slide glass was sealed tightly with an adhesive prepared from coatley plastic powder and Aron Alpha, in the same manner as performed at the time of preparation of CW mouse.
  • the diabetes model mice prepared in Section 16 above were anesthetized with isoflurane using an anesthetizing device for experimental animals (Shinano). Subsequently, the hair in the left half of the back of each mouse was removed with an electric clipper. After the shaven site was disinfected with 70% ethanol, the kidney was exposed by 1.5-2 cm incision. After exposure, the kidney was fixed and the capsule on the ventral side of the kidney was partially incised. Through the resultant opening, three-dimensional tissues prepared in Section 7 above were transplanted. After transplantation, the kidney was returned into the body. Then, the fascia and the skin were sutured.
  • mice which underwent transplantation were anesthetized by ketalar/xylazine mixed anesthesia in the same manner as in Section 11 above.
  • Each mouse was fixed on a 25 ⁇ 60 mm micro cover glass (Matsunami) in the supine position so that the cranial window would become level.
  • Morphological changes of the transplanted three-dimensional tissues with vascular networks were observed with a confocal microscope (LEICA TCS-SP5).
  • mice were anesthetized in the same manner as in Section 15, followed by injection of Alexa-Flour 647 anti-mouse CD31 (Biolegend) antibody at a rate of 100 ⁇ l per 20 g body weight from the tail vein using a 29G syringe. Subsequently, observation was performed in the same manner as described in Section 19 above.
  • mice The internal structure of normal islet tissues was visualized using Pdx-DsRed mice (kindly provided by Mr. Douglous Melton) and CAG-GFP mice (Japan SLC).
  • the mice were anesthetized with isoflurane using an anesthetizing device for experimental animals.
  • the hair on the back of each mouse was removed with an electric clipper.
  • each mouse was incised in the back by 0.5-1 cm so that the spleen was exposed to the outside, whereupon the pancreas adhering in the vicinity of the spleen became exposed.
  • each mouse was held in a 10 cm dish such that the pancreas stuck to the bottom. With each mouse held in this position, 1.5% agarose gel solution cooled to 37° C. was poured into the dish to thereby fix the mouse as the pancreas remained exposed.
  • Normal islet tissues in the fixed mouse were observed with a confocal microscope.
  • a glucose solution 3 g/kg was adjusted with physiological saline to give a dose of 200 ⁇ l per mouse and administered by intraperitoneal injection. After administration, blood samples were taken from the tail vein every 15 min and measured for glucose levels with a Glutest neo SensorTM (Panasonic, Tokyo).
  • Transplanted samples were removed, washed with PBS and fixed in 4% paraformaldehyde for 1 day. Then, the sample tissue was transferred into 10% and 20% sucrose solutions, and kept there until it sank (sucrose replacement). The sinking tissue was transferred from the 20% sucrose solution to a 30% sucrose solution and kept there for 1 day for sucrose replacement.
  • the resultant sample tissue was embedded in O.C.T. compound (Funakoshi Co.), followed by infiltration at 4° C. for 15 min. Subsequently, the sample tissue was mounted on a stand of aluminum foil floating on liquid nitrogen for freezing.
  • the resultant frozen block was sliced thinly into 5 ⁇ m thick sections with a cryostat (Lwica CM1950) and adhered onto a slide glass (Matsunami) Frozen sections were air-dried before use.
  • Transplanted samples were removed, washed with PBS and fixed in 4% PFA for 1 day. After fixation, the sample was washed with PBS three times, and dehydrated with 50, 70, 80, 90, 95 or 100% ethanol for 1 hr at each concentration. After 1 hr dehydration with 100% ethanol, the sample was dehydrated with fresh 100% ethanol for 1 day. The resultant sample was subjected to xylene replacement three times, each for 1 hr and transferred into a thermostat bath for paraffin embedding that was set at 65° C., where the sample was infiltrated with a paraffin:xylene (1:1) mixture for 1 hr and with paraffin three times, each for 2 hr. After infiltration, the sample was embedded in paraffin to prepare a paraffin block.
  • the thus prepared paraffin block was sliced on a microtome thinly into 5 ⁇ m thick sections, which were used as paraffin sections.
  • tissue sections were washed with tap water for 2 min to remove the OCT compound. After washing with deionized water, tissue sections were nuclear-stained with haematoxylin (Wako) for 9 min. Subsequently, the stain solution was washed out with deionized water. The resultant tissue sections were soaked in tap water for 10 min to effect water extraction. Subsequently, after washing with deionized water, the cytoplasm of tissue sections was stained with eosin (Muto Chemical) for 10 min. After removing the excessive eosin with deionized water, tissue sections were dehydrated with a series of ethanol baths at increasing concentrations, cleared with xylene, and shielded.
  • eosin Methoxy Chemical
  • Paraffin sections were infiltrated with 100% xylene three times, each for 5 min and then soaked in 100, 90, 80, 70, 60 or 50% ethanol for 3 min at each concentration to effect deparaffinization that rendered the sections hydrophilic. Subsequently, similar to the frozen sections described above, the hydrophilic sections were washed with deionized water and, thereafter, HE staining was performed.
  • tissue sections were each washed with PBS three times for 5 min and fixed in 4% PFA for 10 min at 4° C. Subsequently, the tissue sections were washed with PBS three times for 5 min, and blocked at 4° C. overnight with a blocking solution containing 10% normal serum of an animal used for secondary antibody preparation (goat). Then, a primary antibody diluted 200-fold with PBS was added and after reaction at 4° C. overnight, the sections were washed with PBS three times for 5 min.
  • a combination of anti-mouse/guinea pig insulin antibody, anti-human/mouse CD31, anti-mouse/rat CD31, anti-human/mouse collagen 4, anti-human/rabbit laminin antibody, and anti-mouse/rabbit caspase-3 antibody was used. Further, a secondary antibody diluted 500-fold with PBS was added to the tissue sections and after reaction at room temperature under shading conditions for 1 hr, the tissue sections were washed with PBS three times for 5 min, shielded with a mounting medium containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen), and observed and photographed with a fluorescence microscope.
  • DAPI 4′,6-diamidino-2-phenylindole dihydrochloride
  • Alexa 488-, 555-labeled goat anti-rabbit IgG (H+L) antibody Alexa 488-, 555-, 647-labeled goat anti-rat IgG (H+L) antibody
  • Alexa 488-, 555-labeled goat anti-guinea pig IgG (H+L) antibody Alexa 488-, 555-, 647-labeled goat anti-mouse IgG (H+L) antibody.
  • Vascularized islets as generated were recovered and fixed in a 4% PFA solution for 1 day, followed by washing with PBS three times for 10 min. After fixation, the islets were placed in a 0.1% Triton-PBS solution containing 3% BSA and blocked at room temperature for 1 hr. After blocking, the islets were washed with a 0.1% Triton-PBS solution three times for 10 min. A transplant was placed in a solution of primary antibody diluted with a 0.1% Triton-PBS solution and reaction was performed at 4° C. for 1 day.
  • the transplant was washed with a 0.1% Triton-PBS solution three times for 10 min and then placed in a solution of secondary antibody diluted with a 0.1% Triton-PBS solution, followed by reaction at room temperature for 4 hr. After the reaction, the transplant was washed with a 0.1% Triton-PBS solution three times for 10 min. A mounting medium containing DAPI was added to the transplant, which was then observed with a confocal microscope.
  • FIG. 1A Media were validated using the survival rate of islet cells as an indicator ( FIG. 1A ). At 72 hours of culture, dead cell numbers per islet area under respective conditions were 14 cells/mm 2 in RPMI1640 medium; 1.8 cells/mm 2 in the mixed medium of RPMI1640 and the endothelial cell medium; and 0.8 cells/mm 2 in the endothelial cell medium ( FIG. 1B ).
  • GFP green fluorescent protein
  • vascularized three-dimensional tissue was autonomously generated by coculturing the three types of cells, i.e., mouse islet, HUVEC and MSC, under appropriate conditions.
  • Mouse islets were cultured as described in Section 10 of Methods above and their survival rates under various conditions were compared ( FIG. 1F , viable cell: green; dead cell: red). At 24 hours of culture, dead cell numbers per islet area under the respective conditions were 53 cells/mm 2 in monoculture of islets alone, 14 cells/mm 2 in coculture with HUVECs, 2 cells/mm 2 in coculture with MSCs, and 0.1 cells/mm 2 in coculture with HUVECs and MSCs ( FIG. 1G ). From these results, it was shown that the survival rate of mouse islet cells was improved by coculturing with HUVECs and MSCs.
  • FIG. 1H culture was performed as described in Section 12 of Methods above and insulin levels secreted from the mouse islets were measured.
  • FIG. 1H insulin levels secreted from the mouse islets were measured.
  • FIG. 1I insulin secretion increased 1.37-fold in the islet monoculture group and 1.97-fold in the coculture group.
  • FIG. 1I changes in gene expressions before and after coculture with HUVCs and MSCs were analyzed comprehensively by microarray analysis.
  • vascularized islet generated in Section 1 of Results above was transplanted into mice and morphological changes in tissues were tracked ( FIG. 3 ). Further, in order to examine the necessity of vascularization for generating tissues, mouse islets alone were transplanted into mice for comparison. Vascularized islets were transplanted into cranial window (CW) mice as described in Section 17 of Methods, and morphological changes were tracked as described in Section 19 of Methods.
  • CW cranial window
  • FIG. 3B After transplantation of mouse islets alone, no macroscopic changes were observed in mouse heads until day 2 post-transplantation. Also, no blood perfusion into transplanted islets was observed. As time passed after transplantation, viable islets decreased ( FIG. 3B ). When fluorescence labeling was used to observe changes in cell morphology, there were no changes, either, but the number of islets gradually decreased. Further, when blood flow was visualized, no blood perfusion into the inside of islets occurred at day 7 post-transplantation ( FIG. 3D , islet: green; blood flow: red). However, in the mouse heads transplanted with vascularized islets, blood perfusion to all over the transplantation site occurred at day 2 post-transplantation ( FIG. 3A ). Further, according to an observation with a confocal microscope, blood perfusion into the inside of islets was confirmed at day 7 post-transplantation ( FIG. 3C , islet: green; blood flow: red).
  • transplantation of vascularized islets induced early resumption of blood flow into the inside of the transplanted islets and improved the islet survival rate after transplantation.
  • FIG. 3E Forty vascularized islets cocultured under the condition of 5 islets were transplanted into the subcapsular space of the kidney of diabetes model mice and evaluated for their therapeutic effects.
  • FIG. 3F Decrease in glucose level was seen at day 1 post-transplantation, and normal glucose level was kept stably retained at week 2 post-transplantation and thereafter ( FIG. 3F ).
  • FIG. 3G Further, a great increase in body weight was seen ( FIG. 3G ) and survival rate improved ( FIG. 3H ).
  • the results of a glucose tolerance test in vivo revealed that the diabetes model mice showed a insulin secretion response which was almost equal to that of normal mice ( FIG. 3I ).
  • FIG. 1E ′ Vascularized islets at day 1 of coculture were analyzed histologically and immunohistologically.
  • islet tissues were observed that had no central necrosis and which adjoined HUVECs and MSCs ( FIG. 2E , upper panel).
  • immunostaining was performed as follows ( FIG. 1E ′; 2 E, lower panel). Briefly, islets were stained with insulin antibodies ( FIG. 1E ′: green; 2 E: red); HUVECs were stained with human vascular endothelial cell antibodies ( FIG. 1E ′: red; 2 E: green); and mouse blood vessels were stained with mouse vascular endothelial cell antibodies ( FIG. 2E : blue). The presence of HUVECs was confirmed in the inside of insulin-positive islets, and HUVECs and mouse blood vessels were connected together.
  • vascularized islets FIG. 3J
  • islets FIG. 3K
  • vascularized islets FIG. 3J
  • islets FIG. 3K
  • HE staining islets engrafting onto the brain tissue were confirmed.
  • immunostaining it was found that human vascular endothelial cells were present at insulin-positive sites in the vascularized islets, and that such human vascular endothelial cells were stable human blood vessels that would secrete laminin and collagen IV (extracellular matrices).
  • islets alone were transplanted, no vascular endothelial cells were found inside the islets.
  • vascularized islets FIG. 3L
  • islets FIG. 3M
  • islets FIG. 3M
  • islets FIG. 3M
  • islets FIG. 3L
  • islets FIG. 3M
  • immunostaining was performed to stain islets (green) with an insulin antibody and vascular endothelial cells (red) with a laminin antibody ( FIG. 3L , lower right panel; FIG. 3M , lower right panel).
  • expression of laminin-positive vascular endothelial cells was confirmed inside insulin-positive islets.
  • no vascular endothelial cells were observed.
  • C57BL/6-Tg mice (Japan SLC, Inc.) anesthetized with diethyl ether (Wako) were laparotomized after disinfection of the abdomen with 70% ethanol.
  • the kidney was cut out and the capsule was removed therefrom. After washing with physiological saline, the kidney was cut in round slices with a scalpel.
  • the renal pelvis and the medulla were removed with scissors, and the cortex was recovered.
  • the recovered cortex was minced on ice and filtered with a 100 ⁇ m mesh cell strainer while adding Hanks' buffer (HBSS, Gibco) containing 0.1% albumin from bovine serum (BSA, Sigma) little by little.
  • HBSS Hanks' buffer
  • BSA bovine serum
  • the flow-through was filtered with a 70 ⁇ m mesh cell strainer, and finally the flow-through was filtered with a 40 ⁇ m mesh cell strainer.
  • the cell mass retained on the 40 ⁇ m mesh cell strainer was recovered with 0.1% BSA-containing Hanks' buffer. The thus recovered material was filtered with a 100 ⁇ m mesh cell strainer.
  • mice glomeruli isolated in Section 1 of Methods above were observed under a stereomicroscope, spherical mouse glomeruli (diameter: 50-100 ⁇ m) could be confirmed. These glomeruli were recovered and transferred to a medium for glomeruli with a Pipetman.
  • Mouse glomeruli were cultured using RPMI1640 (Wako) supplemented with 20% fetal bovine serum (BWT Lot. S-1560), 100 ⁇ g/ml penicillin/streptomycin (Gibco) and Insulin-Transferrin-SeleniumX (Gibco) in a 37° C., 5% CO 2 incubator.
  • HUVECs normal human umbilical vein endothelial cells
  • HUVECs Human umbilical vein endothelial cells
  • hMSCs Human mesenchymal stem cells
  • MSCGMTM BulletKitTM Lonza PT3001
  • Both HUVECs and hMSCs were cultured in a 37° C., 5% CO 2 incubator.
  • mice glomeruli/well were left standing in each well of PrimeSurfaceTM 96-Well U Plate (Sumitomo Bakelite) preliminarily filled with a medium for glomeruli, and 5 ⁇ 10 4 HUVECs and 5 ⁇ 10 3 hMSCs were seeded in each well. Subsequently, the plate was incubated in a 37° C. incubator for one day. Further, 100 mouse glomeruli/well were left standing in each well of a 24-well plate, and 2 ⁇ 10 6 HUVECs and 2 ⁇ 10 5 hMSCs were seeded in each well.
  • PrimeSurfaceTM 96-Well U Plate Suditomo Bakelite
  • Coculture was performed for tracking chronological changes with a stereomicroscope. Briefly, 20 mouse glomeruli/well were left standing in each well of a 24-well plate, and 2 ⁇ 10 6 HUVECs and 2 ⁇ 10 5 hMSCs were seeded in each well. After seeding, the plate was set in a stereomicroscope (Leica DFC300FX) and morphological changes caused by coculture were observed.
  • a stereomicroscope Leica DFC300FX
  • NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used as transplantation animal were bred under a SPF environment with a light-dark cycle consisting of 10 hours for day and 14 hours for night.
  • the breeding of experimental animals were entrusted to the Animal Experiment Center, Joint Research Support Section, Advanced Medical Research Center, Yokohama City University. Animal experiments were performed in accordance with the ethical guidelines stipulated by Yokohama City University.
  • the space between the slide glass and the brain surface was filled with physiological saline and thereafter the slide glass was sealed tightly with an adhesive prepared from coatley plastic powder and Aron Alpha, in the same manner as performed at the time of preparation of CW mice.
  • mice which underwent transplantation were anesthetized by ketalar/xylazine mixed anesthesia in the same manner as in Section 11 above.
  • Each mouse was fixed on a 25 ⁇ 60 mm micro cover glass (Matsunami) in the supine position so that the cranial window would become level.
  • Morphological changes of the transplanted three-dimensional tissues with vascular networks were observed with a confocal microscope (LEICA TCS-SP5).
  • FIGS. 4B and 4D blue
  • HUVECs were found to be scattered evenly around glomeruli.
  • vascularized three-dimensional tissue was autonomously generated by coculturing the three types of cells, i.e. mouse glomeruli, HUVEC and MSC, under appropriate conditions.
  • vascularized glomeruli generated in Section 1 of Results above were transplanted into mice and morphological changes in tissues were tracked ( FIG. 4E ).
  • Vascularized glomeruli were transplanted into cranial window (CW) mice as described in Section 8 of Methods, and morphological changes were tracked as described in Section 9 of Methods.
  • Human pancreatic tumor tissues sliced into 1 mm-square sections were recovered with a Pipetman (20 sections) and mixed with 2 ⁇ 10 6 EGFP-HUVECs and 2 ⁇ 10 5 MSCs. The mixture was centrifuged at 950 rpm. The resultant supernatant was removed, and the cells were suspended in 1 ml of EGM medium and seeded on 24-well plate in which Matrigel was placed in advance. Then, morphological changes were tracked with a confocal microscope.
  • Pancreatic cancer tissues were recovered from pancreatic cancer model mice (Pdx1-cre; LSL-Kras G12D ; CDKN2A ⁇ / ⁇ : purchased from NCI) which are held to be capable of recapitulating the multistep carcinogenesis of pancreatic cancer.
  • the cancer tissues were washed with PBS and transferred to a 6 cm dish containing a HBSS medium under a clean bench environment.
  • the recovered cancer tissues were chopped into 1 mm-square sections, which were used in the subsequent experiments.
  • Pancreatic cancer tissues chopped into 1 mm-square sections were recovered with a Pipetman (20 sections) and mixed with 2 ⁇ 10 6 EGFP-HUVECs and 2 ⁇ 10 5 MSCs. The mixture was centrifuged at 950 rpm for 5 min. The resultant supernatant was removed, and the cells were suspended in 1 ml of EGMTM BulletKitTM (Lonza CC4133) medium and seeded on 24-well plate in which Matrigel was placed in advance. The plate was incubated in a 37° C. incubator for 4 days while exchanging the medium every day.
  • the 24-well plate was prepared as follows. Briefly, 300 ⁇ l of a solution prepared by mixing EGM medium and BD MatrigelTM basement membrane matrix (BD Japan 356234) at 1:1 was added to each well of a 24-well plate, which was then incubated in a 37° C. incubator for 10 min to solidify the gel.
  • pancreatic cancer tissue chopped into 1 mm-square sections was cocultured with HUVECs and MSCs on the MatrigelTM solidified in 24-well plate, vascularized three-dimensional tissues could successfully be generated ( FIG. 5B , upper panel).
  • 1 mm-square sections of the pancreatic cancer tissue alone were cultured on solidified MatrigelTM; neither formation of three-dimensional tissues nor vascularization was confirmed and there occurred no changes worth particular mention ( FIG. 5B , lower panel).
  • cancer stem cells which were conventionally difficult to maintain in vitro—were amplified.
  • Conventional two-dimensional culture systems were difficult to use as a system for pre-evaluating the efficacy of anticancer agents because the two-dimensional system has such an environment that the reactivity of anticancer agents differs greatly from the case where they are administered in vivo.
  • it is expected to reproduce the reactivity in cancer tissues (including vascular systems) in living bodies. This is a culture technique that is potentially highly useful as a drug screening system applicable to the development of novel anticancer agents.
  • C57BL/6-Tg mice Japan SLC, Inc.
  • diethyl ether Wako
  • the liver was cut out, washed with physiological saline and minced with scissors.
  • the minced liver was filtered with a 100 ⁇ m mesh cell strainer while adding Hanks' buffer (HBSS, Gibco) containing 0.1% albumin from bovine serum (BSA, Sigma) little by little.
  • HBSS Hanks' buffer
  • BSA bovine serum
  • Mouse liver tissues were cultured in DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum (ICN Lot. 7219F), 2 mmol/L L-glutamine (Gibco), 100 ⁇ g/mL penicillin/streptomycin (Gibco), 10 mmol/L nicotinamide (Sigma), 50 ⁇ mol/L 2-Mercaptoethanol, 1 ⁇ 10 ⁇ 7 mol/L 6.5% dexamethasone (Sigma), 2.6 ⁇ 10 ⁇ 4 M L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), 5 mmol/L HEPES (DOJINDO), 1 ⁇ g/mL Human recombinant insulin expressed in yeast (Wako), 50 ng/mL Human recombinant HGF expressed in Sf21 insect cells (Sigma) and 20 ng/mL Mouse Submaxillary Glands EGF (Sigma) in a 37° C., 5% CO 2 incubator.
  • HUVECs normal human umbilical vein endothelial cells
  • HUVECs Human umbilical vein endothelial cells
  • hMSCs Human mesenchymal stem cells
  • MSCGMTM BulletKitTM Lonza PT3001
  • Both HUVECs and hMSCs were cultured in a 37° C., 5% CO 2 incubator.
  • NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used as transplantation animal were bred under a SPF environment with a light-dark cycle consisting of 10 hours for day and 14 hours for night.
  • the breeding of experimental animals were entrusted to the Animal Experiment Center, Joint Research Support Section, Advanced Medical Research Center, Yokohama City University. Animal experiments were performed in accordance with the ethical guidelines stipulated by Yokohama City University.
  • mice prepared in Section 8 above underwent transplantation after their brain surfaces were exposed by removing the glass of the cranial window. Those mice which did not have any sign of bleeding, inflammation or infection on their brain surfaces were used. After anesthetization, the area surrounding the cranial window was disinfected with 70% ethanol. The pointed end of an 18G needle was inserted into the border line between the custom-made circular slide glass and Aron Alpha and so manipulated as to peel off the slide glass without damaging the brain surface. Thus, the brain surface was exposed. Subsequently, the brain surface was washed with physiological saline. A tissue transplant was left standing near the center of the brain surface, and the slide glass was remounted.
  • the space between the slide glass and the brain surface was filled with physiological saline and, thereafter, the slide glass was sealed tightly with an adhesive prepared from coatley plastic powder and Aron Alpha, in the same manner as performed at the time of preparation of CW mouse.
  • mice which underwent transplantation were anesthetized by ketalar/xylazine mixed anesthesia in the same manner as in Section 11 above.
  • Each mouse was fixed on a 25 ⁇ 60 mm micro cover glass (Matsunami) in the supine position so that the cranial window would become level.
  • Morphological changes of the transplanted three-dimensional tissues with vascular networks were observed with a confocal microscope (LEICA TCS-SP5).
  • FIG. 6A When mouse liver tissues were cocultured with HUVECs and MSCs, three-dimensional tissues were formed at 24 hours of culture. In order to track morphological changes in cells, coculture experiments were performed using fluorescence-labeled mouse liver tissues and various kinds of cells ( FIG. 6A ). Briefly, liver tissues isolated from mice ( FIG. 6A : red; 6 B, 6 D: green), HUVECs into which green fluorescent protein (GFP) had been introduced (FIG. 6 B) and MSCs were cocultured, followed by observation of cell morphology under a confocal microscope. Immediately after the beginning of culture, HUVECs were confirmed to be scattered evenly around liver tissues.
  • FIG. 6A red; 6 B, 6 D: green
  • FFP green fluorescent protein
  • vascularized three-dimensional tissue was autonomously generated by coculturing the three types of cells, i.e., mouse liver tissue, HUVEC and MSC, under appropriate conditions.
  • the vascularized liver tissues generated in Section 1 of Results above were transplanted into mice, and morphological changes in tissues were tracked ( FIG. 6C ). Transplantation into CW mice was performed as described in Section 6 of Methods, and morphological changes were tracked as described in Section 7 of Methods.
  • mice C57BL/6-Tg mice (Japan SLC, Inc.) anesthetized with diethyl ether (Wako) were laparotomized after disinfection of the abdomen with 70% ethanol.
  • the inlet of the small intestine was cut off by a length of about 20 cm.
  • the lumen of the small intestine thus cut off was washed with 50 ml of physiological saline and then cut lengthwise to expose the mucosa which was cut into small sections of about 5 cm.
  • the resultant small sections were treated in PBS containing 2 mM Ethylenediaminetetraacetic acid (EDTA; Dojinkagaku) and 0.5 mM Dithiothreitol (DTT; Sigma Chemical Company) at 37° C. for 20 min.
  • EDTA Ethylenediaminetetraacetic acid
  • DTT Dithiothreitol
  • the resultant supernatant was passed through a 100 ⁇ m mesh cell strainer and washed with PBS three times.
  • the flow-through was filtered with a 40 ⁇ m mesh cell strainer.
  • the cell mass retained on the 40 ⁇ m mesh cell strainer was recovered with a 0.1% BSA-containing Hanks' buffer.
  • Mouse intestinal tissues were cultured using RPMI1640 (Wako) supplemented with 20% fetal bovine serum (BWT Lot. S-1560), 100 ⁇ g/ml penicillin/streptomycin (Gibco) and Insulin-Transferrin-SeleniumX (Gibco) in a 37° C., 5% CO 2 incubator.
  • HUVECs normal human umbilical vein endothelial cells
  • HUVECs Human umbilical vein endothelial cells
  • hMSCs Human mesenchymal stem cells
  • MSCGMTM BulletKitTM Lonza PT3001
  • Both HUVECs and hMSCs were cultured in a 37° C., 5% CO 2 incubator.
  • mice were left standing in each well of PrimeSurfaceTM 96-well U plate (Sumitomo Bakelite) preliminarily filled with a medium for intestinal tissues. Then, 5 ⁇ 10 4 HUVECs and 5 ⁇ 10 3 hMSCs were seeded in each well. The plate was then incubated in a 37° C. incubator for 1 day.
  • PrimeSurfaceTM 96-well U plate Suditomo Bakelite
  • Coculture was performed for tracking chronological changes with a stereomicroscope. Briefly, mouse intestinal tissues were left standing in each well of a 24-well plate, and 2 ⁇ 10 6 HUVECs and 2 ⁇ 10 5 hMSCs were seeded in each well. After seeding, the plate was set in a stereomicroscope (Leica DFC300FX) and morphological changes caused by coculture were observed.
  • a stereomicroscope Leica DFC300FX
  • NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used as transplantation animal were bred under a SPF environment with a light-dark cycle consisting of 10 hours for day and 14 hours for night.
  • the breeding of experimental animals were entrusted to the Animal Experiment Center, Joint Research Support Section, Advanced Medical Research Center, Yokohama City University. Animal experiments were performed in accordance with the ethical guidelines stipulated by Yokohama City University.
  • the space between the slide glass and the brain surface was filled with physiological saline and, thereafter, the slide glass was sealed tightly with an adhesive prepared from coatley plastic powder and Aron Alpha, in the same manner as performed at the time of preparation of CW mice.
  • mice which underwent transplantation were anesthetized by ketalar/xylazine mixed anesthesia in the same manner as in Section 11 above.
  • Each mouse was fixed on a 25 ⁇ 60 mm micro cover glass (Matsunami) in the supine position so that the cranial window would become level.
  • Morphological changes of the transplanted three-dimensional tissues with vascular networks were observed with a confocal microscope (LEICA TCS-SP5).
  • FIG. 7B vascularized three-dimensional tissues in a culture plate (substrate?) of such a shape that cells/tissues would gather in the bottom.
  • FIG. 7B fluorescence-labeled mouse intestinal tissues were cocultured with various kinds of cells.
  • FIG. 7B intestinal tissues isolated from mice ( FIG. 7B : red), HUVECs into which green fluorescent protein (GFP) had been introduced ( FIG. 7B ) and MSCs were cocultured, and cell morphology was observed with a confocal microscope Immediately after the beginning of culture, HUVECs were confirmed to be scattered evenly around intestinal tissues.
  • GFP green fluorescent protein
  • vascularized three-dimensional tissue was autonomously generated by coculturing the three types of cells, i.e., mouse intestinal tissue, HUVEC and MSC, under appropriate conditions.
  • vascularized intestinal tissues generated in Section 1 of Results above were transplanted into mice and morphological changes in tissue were tracked ( FIG. 7C ).
  • vascularized intestinal tissues were transplanted into cranial window (CW) mice as described in Section 6 of Methods, and morphological changes were tracked as described in Section 7 of Methods.
  • FIG. 7C In the mouse heads transplanted with vascularized intestinal tissues, blood perfusion to all over the transplantation site occurred at day 3 post-transplantation. Further, observation with a confocal microscope confirmed that blood perfusion into the inside of the transplanted intestinal tissues occurred at day 3 post-transplantation. ( FIG. 7D ).
  • C57BL/6-Tg mice Japan SLC, Inc.
  • diethyl ether Wako
  • the lungs were washed with physiological saline and minced with scissors.
  • the minced lung was filtered with a 100 ⁇ m mesh cell strainer while adding Hanks' buffer (HBSS, Gibco) containing 0.1% albumin from bovine serum (BSA, Sigma) little by little.
  • the flow-through was filtered with a 40 ⁇ m mesh cell strainer.
  • the cell mass retained on the 40 ⁇ m mesh cell strainer was recovered with a 0.1% BSA-containing Hanks' buffer.
  • HUVECs normal human umbilical vein endothelial cells
  • HUVECs Human umbilical vein endothelial cells
  • hMSCs Human mesenchymal stem cells
  • MSCGMTM BulletKitTM Lonza PT3001
  • Both HUVECs and hMSCs were cultured in a 37° C., 5% CO 2 incubator.
  • mice pulmonary tissues were left standing in each well of PrimeSurfaceTM 96-well U plate (Sumitomo Bakelite) filled with a medium for pulmonary tissues. Then, 5 ⁇ 10 4 HUVECs and 5 ⁇ 10 3 hMSCs were seeded in each well. The plate was then incubated in a 37° C. incubator for 1 day. Further, mouse pulmonary tissues were left standing in each well of a 24-well plate. Then, 2 ⁇ 10 6 HUVECs and 2 ⁇ 10 5 hMSCs were seeded in each well.
  • Coculture was performed for tracking chronological changes with a stereomicroscope. Briefly, 20 mouse pulmonary tissues were left standing in each well of a 24-well plate. HUVECs (2 ⁇ 10 6 cells) and hMSCs (2 ⁇ 10 5 cells) were seeded in each well. After seeding, the plate was set in a stereomicroscope (Leica DFC300FX) and morphological changes caused by coculture were observed.
  • a stereomicroscope Leica DFC300FX
  • NOD/SCID mice (Sankyo Labo Service Co., Tokyo, Japan) used as transplantation animal were bred under a SPF environment with a light-dark cycle consisting of 10 hours for day and 14 hours for night.
  • the breeding of experimental animals were entrusted to the Animal Experiment Center, Joint Research Support Section, Advanced Medical Research Center, Yokohama City University. Animal experiments were performed in accordance with the ethical guidelines stipulated by Yokohama City University.
  • mice prepared in Section 8 underwent transplantation after their brain surfaces were exposed by removing the glass of the cranial window. Those mice which did not have any sign of bleeding, inflammation or infection on their brain surfaces were used. After anesthetization, the area surrounding the cranial window was disinfected with 70% ethanol. The pointed end of an 18G needle was inserted into the border line between the custom-made circular slide glass and Aron Alpha and so manipulated as to peel off the slide glass without damaging the brain surface. Thus, the brain surface was exposed. Subsequently, the brain surface was washed with physiological saline. A tissue transplant was left standing near the center of the brain surface, and the slide glass was remounted.
  • the space between the slide glass and the brain surface was filled with physiological saline and, thereafter, the slide glass was sealed tightly with an adhesive prepared from coatley plastic powder and Aron Alpha, in the same manner as performed at the time of preparation of CW mouse.
  • mice which underwent transplantation were anesthetized by ketalar/xylazine mixed anesthesia in the same manner as in Section 11 above.
  • Each mouse was fixed on a 25 ⁇ 60 mm micro cover glass (Matsunami) in the supine position so that the cranial window would become level.
  • Morphological changes of the transplanted three-dimensional tissues with vascular networks were observed with a confocal microscope (LEICA TCS-SP5).
  • FIG. 8A fluorescence-labeled mouse pulmonary tissues and various kinds of cells. Briefly, pulmonary tissues isolated from mice ( FIG. 8A : red), HUVECs into which green fluorescent protein (GFP) had been introduced ( FIG. 8A : green) and MSC were cocultured, followed by observation of cell morphology under a confocal microscope. Immediately after the beginning of culture, HUVECs were confirmed to be scattered evenly around pulmonary tissues.
  • GFP green fluorescent protein
  • vascularized three-dimensional tissue was autonomously generated by coculturing the three types of cells, i.e., mouse pulmonary tissue, HUVEC and MSC, under appropriate conditions.
  • the vascularized pulmonary tissues generated in Section 1 of Results above were transplanted into mice, and morphological changes in tissues were tracked ( FIG. 8B ).
  • Transplantation into CW mice was performed as described in Section 16 of Methods, and morphological changes were tracked as described in Section 7 of Methods.
  • Expanded but undifferentiated iPS cells (kindly provided by Dr. Nakauchi, Tokyo University; TkDA3 clone; established from dermal fibroblasts) were washed once with a washing medium (DMEM/F12; Life Technologies 11320).
  • a cultured cell dissociating solution (Funakoshi AT104) was added to 100 mm dishes in an amount of 1 ml per dish. Cells were recovered into 50 ml centrifugal tubes and subjected to centrifugation at 900 rpm for 5 min. After taking a cell count, cells were seeded on MatrigelTM-coated 60 mm dishes at a density of 1.5 ⁇ 10 6 cells per dish. MatrigelTM-coating was performed as follows.
  • BD MatrigelTM basement membrane matrix (BD Japan 356231) was diluted 30-fold with DMEM (Life Technologies 1196118). The thus diluted gel was added to 60 mm dishes (2 ml/dish), which were left standing at room temperature for 2 hr.
  • a culture broth an iPS culture medium supplemented with ROCK inhibitor Y-27632 (Calbiochem 688000) was used. Cells were incubated in a 37° C. incubator for 24 hr to induce cell adhesion. Subsequently, the culture broth was exchanged with a directed differentiation medium.
  • This medium was RPMI-1640 (Wako Pure Chemicals 189-02025) supplemented with B-27TM Supplement Minus Insulin (Life Technologies 0050129SA) (1/100 dilution) and 100 ng/ ⁇ l Activin A (Ajinomoto). While exchanging the medium every 2 days, cells were cultured for 6 days to allow directed differentiation into definitive endoderms. The degree of differentiation into endodermal lineage was confirmed by quantitative PCR and immunostaining.
  • EZSPHERETM Human iPS cells which had undergone directed differentiation into definitive endoderms were seeded in each well of EZSPHERETM (Asahi Glass 4810-900 6-well-Flat bottom) at a density of 1.0 ⁇ 10 6 cells/well.
  • a culture broth As a culture broth, a 1:1 mixture of a medium kit for sole use with hepatocytes (HCMTM BulletKitTM; Lonza CC3198) and EGMTM BulletKitTM (Lonza CC-4133) was used.
  • HCMTM BulletKitTM hepatocytes
  • EGMTM BulletKitTM Lonza CC-4133
  • iPS cell-derived endodermal tissues were left standing in each well of PrimeSurfaceTM 96-Well U Plate (Sumitomo Bakelite) preliminarily filled with the medium for culturing iPS cell-derived endodermal tissues described in Section 2 above. Then, 1.0 ⁇ 10 4 HUVECs and 1.0 ⁇ 10 3 hMSCs were seeded in each well. Subsequently, the cells were incubated in a 37° C. incubator for 4 days.
  • PrimeSurfaceTM 96-Well U Plate Suditomo Bakelite
  • Bio tissues integrated with a vascular system according to the present invention are applicable to generation of human functional cells, organ transplantation, drug discovery screening, new analytical systems for evaluating such factors as the relationship between development of drug efficacy and blood vessels.

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