Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Culture systems for three-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated two methods to enhance oxygen supply: (1) using a culture plate with a gas-permeable polydimethylsiloxane (PDMS) membrane on the bottom, and; (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids, comprised of 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads, did not develop a necrotic core for nine days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

DOI： 10.3390/cells8060525

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DOI： 10.1016/j.toxlet.2018.10.023

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DOI： 10.1007/978-1-4939-8961-4_14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

Force sensing is one of the key challenges in realizing mechanical characterizations of living samples. Since living samples are normally cultured within a medium and tend to have nonlinearity in terms of their strain-stress curve, the two important features; a wide measurement range and being waterproof are required for the force sensor. Given these key requirements, in this paper, we present a force sensor probe that uses a quartz crystal resonator (QCR) to achieve the mechanical characterization of a HepG2 spheroid. The force sensor probe was fabricated using five-layered structure of quartz wafers; here, the layers are packaged using atomic diffusion bonding. We evaluated the performance of the fabricated force sensor probe, with results showing that the sensor had a wide measurement range of 1.8 x 10(6) from 0.19 Nto 340 mN. By using a constructed mechanical characterization system integrated with the QCR force sensor probe, we evaluated the stiffness of HepG2 spheroids over different culture periods. To evaluate the stiffness as a mechanical characteristics of HepG2 spheroid, we introduced an index Sistope. The results of mechanical characterization indicated that the measured Sistope changed along with each culture day. From these results, we succeeded in measuring mechanical characteristics of spheroids using our fabricated force sensor probe. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.sna.2017.08.033

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Introduction: Three-dimensional (3D) multicellular spheroids are useful tools for simulation of cellular functions in vitro. However, it is difficult to culture certain epithelial cell types under 3D spheroid conditions because these cells cannot resist autonomous cell death, triggered by disordered cell polarity. The objective of this study was to find a method that enables spheroid culture of such epithelial cells utilizing hydrogel beads without cell death.
Methods: We used murine E14.5 fetal hepatic cells for the spheroid composition because they are sensitive to disorganized structures. Spheroids were formed by injecting 1-mu l fresh medium containing 1000 fetal hepatic cells and the same number of alginate hydrogel beads (20 mm in diameter) into a 3% methylcellulose medium in the presence of dexamethasone and oncostatin M to induce hepatic differentiation. After 7 days of culture, microstructures were observed using hematoxylin and eosin staining and immunostaining using anti-CK8/18 antibody. Albumin secretion rate was determined by the enzyme-linked immunosorbent assay method. In addition, polarity-related proteins, E-cadherin, ezrin, and MRP2 were observed with immunostaining.
Results: Control spheroids without the use of alginate hydrogel beads showed extensive internal lack of epithelial hepatic cells. The spheroids containing alginate hydrogel beads exhibited sheet-or cord-like structures of epithelial hepatic cells, and it was clear that cell death of epithelial cells had been prevented. Albumin secretion data also supported the improvement of epithelial hepatic cell viability when alginate hydrogel beads were used. Localization of polarity-related proteins indicated the partial reconstitution of cell polarity in the spheroids using alginate hydrogel beads.
Conclusion: Based on these data, we concluded that the application of alginate hydrogel beads was effective in improving the epithelial hepatic cell culture of multicellular spheroids. (C) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.reth.2016.01.007

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We have previously reported that multi-cellular heteroaggregates comprising murine pancreatic alpha (alpha TC1.6) and beta (MIN6-m9) cell lines spontaneously acquired islet-like architecture and displayed higher insulin secretion rates. However, the mechanisms of self-organization remain unclear. The objective of this study is to examine the possibility that a sugar chain participates in the mutual recognition of the cells during reconstitution of the islet-like structure in vitro. Using a lectin-binding assay, we identified Erythrina cristagalli agglutinin (ECA), which particularly recognizes the beta-galactoside structure on the surfaces of MIN6-m9 cells. The self-organization of alpha TC1.6 and MIN6-m9 was obstructed using ECA-bound MIN6-m9 cells. Lactose neutralized the ECA's inhibitory effect on the autonomous rearrangement of alpha TC1.6 and MIN6-m9 cells, indicating that the inhibition of cell arrangement by ECA was mediated via b-galactoside. We concluded that a beta-galactoside sugar chain was central to the reconstitution of the pancreatic islet-like architecture in vitro. (C) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.reth.2016.01.006

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T-box transcription factors play important roles in vertebrate mesoderm formation. Eomesodermin is involved in the initial step of the prospective mesodermal cells recruited near the primitive streak. Then T or Brachyury gene is responsible for general and axial mesodermal development. Tbx6, on the other hand, promotes paraxial mesodermal development while suppressing neural differentiation. Here, we studied differentiative properties of mouse ES cells (mESCs) with its Tbx6 expression regulated under the Tet-off system. mESCs were treated with noggin to promote neural differentiation. When Tbx6 was simultaneously turned on, later neural differentiation of these cells hardly occurred. Next, mESCs were subjected to formation of the embryoid bodies (EBs). When Tbx6 was turned on during EB formation, the rate of later cardiac troponin T (cTnT)-positive cells increased. If the cells were further treated with a wnt inhibitor KY02111 after EB formation, a synergistic increase of cTnT-positive cells occurred. Tbx6 expression in mESCs influenced the constituent ratio of the cardiac myosin light chain types, such that atrial species markedly increased over ventricular ones. These results are coincident with the function of Tbx6 in normal development, in that Tbx6 strongly suppressed neural differentiation while promoting cardiac development in a cooperative manner with wnt inhibition. (C) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.reth.2016.02.001

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The regulation of Sertoli cells by some hormones and signaling factors is important for normal spermatogenesis. Notch signaling is considered to be necessary for normal spermatogenesis in mouse. In this study, we revealed two new facts about Sertoli cells by western blotting experiments on different types of primary cells and microdissected tubules. The first is that Sertoli cells express the Jagged1 ligand in mice testes. The second is that the expression level of Jagged1 oscillates in the seminiferous epithelial cycle. Therefore, we inferred that Jagged1 in Sertoli cells contributes to the Notch signaling involved in spermatogenesis. Furthermore, we examined the regulation of Jagged1 expression and found that Jagged1 expression was suppressed by cAMP signaling and was promoted by TNF-alpha signaling in Sertoli cells. When cAMP and TNF-alpha were simultaneously added to Sertoli cells, Jagged1 expression was suppressed. Therefore, cAMP signaling dominates Jagged1 expression over TNF-alpha signaling. These results suggest that cAMP signaling may cause the periodicity of Jagged1 expression in the seminiferous epithelial cycle, and controlling Jagged1 expression by adding TNF-alpha or cAMP may contribute to normal spermatogenesis in vitro. (c) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.reth.2016.02.005

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A three-dimensional (3D) bone marrow (BM) culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC) medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFR alpha, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue. (c) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

DOI： 10.1016/j.reth.2016.01.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Gal1-4Gal1-4Glc). MytiLec revealed -trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)- (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF- production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

DOI： 10.3390/md13127071

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

Multicellular spheroids are indispensale in cell biology as three-dimensional environments, in tissue engineering for achieving higher functions, and in drug validation by pharmaceutical companies. However, spheroids have limited nutrient exchange because they lack microvasulature. In this study we established a method for fabricating microchannel networks in multicellular spheroids to overcome this limitation. Alginate hydrogel beads with almost the same size as animal cells were prepared by discharging alginate solution using an inkjet system system. Using these beads, we produced ''heterospheoids'' that comprised the same number of cells and hydrogel beads based on the rapid cell/particle aggregation method. The hydrogel beads occupied the branched, connected spaces in the heterospheroids, which resembled microvasulature. The connectivity of the microchannel networks was confirmed using confocal laser microscopy and staining cells with 1-mu m polystyrene beads after digesting the hydrogel beads using alginate lyase solution. The microchannel networks improved the albumin secretion rate and supressed the expression of hypoxia-inducible factor-1 alpha in Help G2 cells. Experiments with rat primary hepatocytes demonstrated that the branched luminal-like structures overcame the limitations of albumin secretion and ammonia clearance. These findings suggest that it is possible to fabricate microchannel networks that can effectively maintain cellular fucntions by enhancing material exchange. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2014.02.099

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Engineered pseudoislets reconstituted from a suspension of pancreatic alpha and beta cells have the potential to relieve the shortage of donor islets for transplantation in the treatment of type 1 diabetes. However, the methods to fabricate pseudoislets are not well developed. In this study, we attempted to generate pseudoislets, which show a higher potential for glucose-induced insulin secretion, by altering total cell number, adjusting the cell ratio of pancreatic alpha and beta cells, and fabricating microchannel networks with the use of alginate hydrogel beads. To effectively aggregate alpha and beta cells and hydrogel beads, we used a previously established rapid aggregation method. When pseudoislets were reconstituted with 8,000 cells in a 1:8 alpha/beta-cell ratio, we observed that the glucose-induced insulin secretion was enhanced by 3.1 times compared with the pseudoislets formed with beta cells only. In addition, embedding of microchannel networks increased the insulin secretion rate by 4.4 times compared with the pseudoislets without the microstructures. These findings demonstrated that active modification was effective in reconstituting higher functional pseudoislets, which may be useful for islet transplantation.

DOI： 10.1016/j.transproceed.2013.11.147

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：TAYLOR & FRANCIS INC  

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.

DOI： 10.4161/org.28351

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Our objective is to control the reconstitution of liverlike tissues at extrahepatic sites using hepatic progenitor cells (HPCs) and in vitro preculture prior to transplantation. We prepared cell-based hybrid grafts by culturing HPCs isolated from fetal E14.5 mouse livers on biodegradable, highly porous 3-dimensional poly- L-lactic acid (PLLA) scaffolds for 1 week in basal medium (the basal condition) or 10 mM nicotinamide (NA) and 1% dimethyl sulfoxide (DMSO) supplemented conditions (the ND-positive condition) prior to implantation. Sections of hybrid grafts cultured for 1 week showed that HPCs grew and spread on the surface of scaffolds under both basal and ND (+) conditions. Most of these cells were albumin (+) and CK18 (+). CK19 (+) cells were also present under the basal condition but not the ND (+) condition. Cultured hybrid grafts were implanted into the mesenteric leaves of mice and removed after 1 month. Transplanted tissues cultured under the basal condition consisted of albumin (+) hepatocyte-like and CK19 (+) biliary epithelial cell (BEC)-like cells organized in duct-like structures. In contrast, integrated tissues cultured under the ND (+) condition alone had differentiated albumin (+) hepatocyte-like cells and were relatively larger than those under the basal condition. Hepatocyte-like cells of transplanted hybrid grafts cultured under both conditions were periodic acid-Schiff (PAS) staining-positive and expressed transcription factors, hepatocyte nuclear factor (HNF) 4 and CCAAT/enhancer-binding protein (C/EBP) α. These findings suggest that combining progenitor cells and in vitro preculture may potentially regulate liverlike tissues at extrahepatic sites.

DOI： 10.20965/jrm.2013.p0698

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Mary Ann Liebert Inc.  

One of the most challenging issues in liver tissue engineering is the incorporation of a functional bile duct (BD) network in the hepatic tissue. In this study, we investigated the feasibility of a three-dimensional (3D) coculture of adult rat primary biliary epithelial cells (ABECs) and rat fetal liver cells (FLCs) to form functional BD structures. 3D hetero-spheroids containing various ratios of the two cell types were successfully obtained in low-adherence, round-bottom, 96-well plates. Histological analyses and functional analysis using fluorescein diacetate demonstrated that the ABECs critically contributed to the reconstruction of continuous ductular networks. Metabolized fluorescein was transported toward cystic structures in a time- and ABEC ratio-dependent manner. In particular, the duct-like structures containing fluorescein accumulations interlinked the large cystic accumulations. Furthermore, transplantation experiments demonstrated that ABECs, but not FLCs, enabled the establishment of BD networks in vivo. This study provided important insights into the development of transplantable liver tissues with bile excretion functionality. © Mary Ann Liebert, Inc.

DOI： 10.1089/ten.tea.2013.0021

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We report a method for the rapid production of cellular aggregates without electric power and cell modification. We focused on the swelling property of a solution containing a high molecular material, methylcellulose (MC), which immediately absorbs a small amount of solvent and fills the space occupied by the solvent. When 1 mu l of a suspension of 1000 animal cells in normal culture medium was injected into the 3% MC medium, the normal medium was rapidly absorbed by the surrounding MC medium. Suspended cells were simultaneously trapped on the interfaces between the normal and MC media; they were finally pulled together and held in the MC medium. This event was nearly complete within the first 10 min. Moreover, MC medium-dependent aggregation was observed when polystyrene microspheres of different sizes (diameter, 100 nm-100 mu m) were added. Furthermore, we demonstrated the stepwise fabrication of multi-layered aggregates with embedded structures. These methods for creating engineered aggregates should enhance the study of three-dimensional cultures comprising two or more cell types with well-designed structures. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2012.02.065

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt. we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.12.071

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP PUBLISHING LTD  

Implantation of sheet-like liver tissues is a promising method in hepatocyte-based therapies, because angiogenesis is expected to occur upon implantation from the surrounding tissues. In this context, we introduce here a new methodology for the formation of a functional thick hepatic tissue usable for cell sheet technology. First, we report the formation of composite tissue elements in suspension culture. Composite elements were composed of human hepatoma Hep G2 cells and mouse NIH/3T3 fibroblasts which are important modulators for thick-tissue formation. To overcome the very low attachment and organization capability between different cells in suspension, we synthesized a new cell-to-cell binding molecule based on the avidin-biotin binding system that we previously applied to attach hepatocytes on artificial substrata. This newly synthesized biotin-conjugated biocompatible anchoring molecule was inserted in the plasma membrane of both cell types. NIH/3T3 cells were further conjugated with avidin and incubated with biotin-presenting Hep G2 cells to form highly composite tissue elements. Then, we seeded those elements on highly gas-permeable membranes at their closest packing density to induce the formation of a thick, composite, functional hepatic tissue without any perfusion. This methodology could open a new way to engineer implantable thick liver tissue sheets where different cell types are spatially organized and well supplied with oxygen.

DOI： 10.1088/1758-5082/3/3/034111

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Multicellular heterospheroids including two or more cell types have some tissue/organ properties and can be used in cell-to-cell interaction studies. However, the spheroid formation is difficult to control because the adhesion efficacy is different in each cell type. To solve this, we applied a rapid cell-to-cell adhesion method, avidin biotin (AB) binding, to spheroid formation. Introduction of avidin or biotin molecules to the cell surfaces of Mile Sven 1 (MS1) cells promoted formation of spheroid in minutes. This method allowed the construction of heterospheroids having homogenous distributions of different cell types. Interestingly, cells showed self-organization and MS1 cells formed networks with Hep G2 cells. NIH3T3 cells also remodeled when mixed with Hep G2 cells. In contrast, a combination of MS1 and NIH3T3 cells failed to show pattern formation, indicating that self-organization was based on the composition of cell types. Actin polymerization not cell proliferation was the dominant factor in remodeling of heterospheroids in the first 24 h. We also demonstrated the self-organization of spheroids comprising three different cell types. The new technology to assemble cells is important not only to study cell-to-cell interaction but also to make three-dimensional complicated tissues. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2011.04.081

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

We found in our present study that lithium (Li+) induced the expression of endogenous c-Ret, a tyrosine kinase receptor, in murine inner medullary collecting duct (mIMCD-3) cells. Delineation of the promoter region required for the effect of Li+ identified a positive regulatory element within 180 bp upstream of the transcription initiation site. This region contained three putative GC-rich Sp1 binding sites found to be essential for c-Ret induction by Li+. The effect of Li+ was mediated through glycogen synthase kinase 3 beta (GSK-3 beta) inhibition, although there was no biding site for T cell factor/lymphoid enhancer factor (TCF/LEF) in the 180 bp. We found that Li+ activated the mammalian target of rapamycin (mTOR) pathway via GSK-3 beta in these cells, and the effect of Li+ to induce c-Ret was amenable to the inhibitory effect of the mTOR inhibitor. rapamycin. We also found that alterations in both cellular beta-catenin levels and mTOR activities affected the effect of Li+ on c-Ret transcription in a cooperative manner. In summary, our results show that Li+ can induce c-Ret expression in mIMCD-3 cells through both beta-catenin- and mTOR-dependent pathways downstream of GSK-3 beta, inhibition, which act synergistically on the GC-rich Sp1 binding elements in the promoter region. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellsig.2010.10.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Pluripotent stem cells are under the influence of soluble factors in a diffusion dominant in vivo microenvironment. In order to investigate the effects of secreted soluble factors on embryonic stem cell (ESC) behavior in a diffusion dominant microenvironment, we cultured mouse ESCs (mESCs) in a membrane-based two-chambered micro-bioreactor (MB). To avoid disturbing the cellular environment in the top chamber of the MB, only the culture medium of the bottom chamber was exchanged. Cell growth in the MB after 5 days of culture was similar to that in conventional 6-well plate (6-WP) and membrane-based Transwell insert (TW) cultures, indicating adequate nutrient supply in the MB. However, the cells retained higher expression of pluripotency markers (Oct4, Sox2 and Rex1) and secreted soluble factors (FGF4 and BMP4) in the MB. Inhibition of FGF4 activity in the MB and TW resulted in a similar cellular response. However, in contrast to the TW, inhibition of BMP4 activity revealed that autocrine action of the upregulated BMP4, which acted cooperatively with leukemia inhibitory factor (LIF), upregulated the pluripotency markers expression in the MB culture. We propose that BMP4 accumulated in the diffusion dominant microenvironment of the MB upregulated its own expression by a positive feedback mechanism-in contrast to the macro-scale culture systems-thereby enhancing the pluripotency of mESCs. The micro-scale culture platform developed in this study enables the investigation of the effects of soluble factors on ESCs in a diffusion dominant microenvironment, and is expected to be used to modulate the ESC fate choices.

DOI： 10.1007/s10544-010-9464-8

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DOI： 10.20965/jrm.2010.p0619

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

A reconstituted porous elastin film prepared by a novel cross-linker, Dode-DSP. was examined for its feasibility as a biocompatible and elastic cellular scaffold. Although the mechanical properties of the film alone drastically deteriorated in physical conditions due to degradation, loading of fibroblasts retarded the deterioration in terms of distensibility and E value (Young's modulus).

DOI： 10.1246/cl.2009.878

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

The highly oxygen-permeable material, polydimethylsiloxane (PDMS), has the potential to be applied to cell culture microdevices,"but cell detachment from PDMS has been a major problem. In this study, we demonstrate that a combination of collagen covalently immobilized PDMS and an adequate oxygen supply enables the establishment of a stable, attached spheroid (hemispheroid) culture of rat hepatocytes. The bottom PDMS surfaces were first treated with oxygen plasma, then coupled with aminosilane followed by a pbotoreactive crosslinker, and they were finally reacted with a collagen solution. X-ray photoelectron spectroscopy (XPS) and contact angle measurements showed that the covalent immobilization of collagen on the surface occurred only where the crosslinker had been introduced. On the collagen-conjugated PDMS surface, rat hepatocytes organized themselves into hemispheroids and maintained the viability and a remarkably high albumin production at least for 2 weeks of culture. In contrast, hepatocytes on the other types of PDMS surfaces formed suspended spheroids that had low albumin production. In addition, we showed that blocking the oxygen supply through the bottom PDMS surface inhibited the formation of hemispheroids and the augmentation of hepatocellular function. These results show that appropriate surface modification of PDMS is a promising approach towards the development of liver tissue microdevices.

DOI： 10.1002/bit.21690

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In in vivo liver tissue, each hepatocyte has intimate interactions not only with adjacent hepatocytes but also with nonparenchymal cells in a three-dimensional (3D) manner. We recently reported that hepatic function is highly maintained on collagen covalently immobilized polydimethylsiloxane (PDMS) membranes through which oxygen is supplied directly to the cells. In this study, to further enhance performances of hepatocytes culture, we investigated cocultivation of rat hepatocytes with a mouse fibroblast, NIH/3T3 (M) in the same PDMS membranes. Various functions of hepatocytes were better maintained on the membrane at remarkably higher levels, particularly albumin secretion on such coculture was about 20 times higher than that in conventional coculture on tissue-culture-treated polystyrene (TCPS) surfaces. The remarkable functional enhancements are likely to be explained by the net growth of hepatocytes (from 1.2- to 1.4-fold inoculated number) and very intimate contact between hepatocytes and 3T3 cells in almost continuous double-layered structures under the adequate oxygen supply. The results demonstrate that simultaneous realization of different requirements toward mimicking in vivo liver tissue microstructure is effective in improving performance of hepatocytes culture system. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2007.08.041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

Fetal liver cell fractions are potent sources of cells for future liver tissue engineering, by virtue of their high proliferation capacity and their potential for hepatic maturation. Recently, some types of hepatic differentiation agents have been identified from findings in stem cell biology. We therefore investigated the in vitro growth and maturation of rat fetal liver cells isolated from 17-day-old pregnant rats in poly-L-lactic acid three-dimensional (3D) macroporous scaffolds in the presence of soluble factors, such as a combination of hepatocyte growth factor, fibroblast growth factor-1, and fibroblast growth factor-4, oncostatin M, and sodium butyrate. Inclusion of all these factors in the 3D culture induced higher levels of hepatic functions and well maintained these enhanced levels during 2 weeks of culture, whereas in the monolayer culture, such functional enhancement was gradually lost after 1 week. The finally achieved functions on a per-cell basis in the 3D culture with all of the soluble factors were comparable to those of adult hepatocytes. We therefore conclude that the 3D culture system shows promise for the in vitro maturation of fetal liver cells as a means of preconditioning of the cells for engineered liver tissue equivalents in future transplantation studies.

DOI： 10.1089/ten.a.2007.0079

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

DOI： 10.1263/jbb.104.343

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

To engineer implantable liver tissues, we designed a novel scaffold with a three-dimensional (31)) branching and joining flow-channel network comprising multiple tetrahedral units (4-mm edge length). For the fabrication of this network, biodegradable polycaprolactone (PCL) and 80% (w/w) NaCl salt particles serving as porogen were thoroughly mixed and applied in a selective laser sintering (SLS) process, a technique adapted to rapid prototyping. We thus obtained a scaffold that had high (89%) porosity with a pore size of 100-200 mu m and 3D flow channels. To evaluate its biocompatibility, human hepatoma Hep G2 cells were seeded into the scaffold using avidin-biotin (AB) binding and cultured in a perfusion system for 9 days. The results demonstrated that such 3D flow channels are essential to the cells' growth and function. In addition, the AB binding-based seeding remarkably improved the overall performance of the cell-loaded scaffolds. The fabrication of a much finer scaffold, having a 500cm(3) scale, based on the same design and the use of human hepatocyte progenitors, may, in the near future, lead to the development of an implantable liver tissue equivalent for use in humans. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2007.05.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

C/EBP alpha is a key transcription factor indispensable for the onset of gluconeogenesis in perinatal liver. However, C/EBP alpha was already expressed in fetal liver, suggesting that the expression of C/EBP alpha alone does not account for the dramatic increase of the expression of metabolic genes, and hence an additional factor(s) is expected to function cooperatively with C/EBP alpha in perinatal liver. We show here that expression of Foxo1 was sharply increased in the perinatal liver and augmented C/EBP alpha-dependent transcription. Foxo1 bound C/EBP alpha via its forkhead domain, and Foxo1 bound to the promoter of a gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK), in a C/ EBP alpha-dependent manner in vivo. Insulin inhibited the expression of PEPCK in a culture of fetal liver cells, and also the C/EBP alpha-dependent transcription enhanced by Foxo1. These results indicate that Foxo1 regulates gluconeogenesis cooperatively with C/EBP alpha, and also links insulin signaling to C/EBP alpha during liver development.

DOI： 10.1038/sj.emboj.7601784

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Efficient cell attachment to biodegradable polymer scaffolds is a necessary prerequisite in tissue engineering. However, it is difficult to evenly cover scaffold surfaces with cells because scaffolds are generally highly porous, with complex three-dimensional (3D) surfaces. In this article, we demonstrate the efficiency of avidin-biotin binding systems (ABBS) for the initial attachment of biotinylated Hep G2 cells to avidin adsorbed flat, two-dimensional (2D) and highly porous 3D Poly L-lactic acid (PLLA) surfaces. The potential toxicity of biotinylation and/or strong ABBS binding forces was also investigated. ABBS assisted Hep G2 cells to adhere to a flat PLLA surface within 10 min; the proliferation of these attached cells was comparable with control intact cells cultured on collagen. Hepatic functions of the attached cells, such as albumin secretion, induction of CYP1A1 and CYP1A2 genes, and metabolic capacity of CYP1A1/2 as measured by the ethoxyresorufin O-deethylase assay, were not significantly changed. Also, a stimulus of a cytokine: oncostatin M (OSM) phosphorylated an intracellular signaling molecule, extracellular signal-related kinase I (ERK1) via transmembrane receptor complex, at 24 h after inoculation by ABBS. In addition, efficient attachment of Hep G2 cells to a highly porous PLLA 3D scaffold was demonstrated. These results clearly show that ABBS is useful for rapidly trapping cells in both biodegradable, polymer-based, flat 2D surfaces, and in highly porous 3D scaffolds. Furthermore, binding hepatic cells by this technique has only small effects on liver-specific functions, or on signal transfer ability of transmembrane receptor complexes. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2006.05.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COGNIZANT COMMUNICATION CORP  

To engineer liver tissues with a clinically significant size, in vivo evaluation of performance using largescale animal studies are necessary before proceeding to human clinical trials. As pigs are the most suitable candidates, the development of culture conditions suitable for porcine hepatocyte progenitors is very important to engineer pig liver tissue equivalents. We therefore investigated the efficacy of poly-L-lactic acid (PLLA) three-dimensional (3D) scaffolds on the functional maturation of fetal porcine hepatocytes in the presence of various combinations of biofactors. Cells were isolated from pig fetuses obtained from a local slaughterhouse, and cultured for 15 days both in monolayer and PLLA scaffolds. Although 15 days of culture resulted in almost the same ratio of proliferation (about fivefold) in both monolayer and 3D PLLA culture, the PLLA culture with hepatocyte growth factor (HGF, 10 ng/ml) and sodium butylate (Sb, 1 mM) remarkably enhanced various liver-specific functions of fetal porcine hepatocytes. The final attained functions based on the numbers of immobilized cells on day 1 compared with those of day 1 monolayers; 191-fold increase in albumin secretion, 70.5-fold increase in cytochrome P450 IA1/2 capacity, 20.9-fold increase in ammonia removal, and 18.0-fold increase in urea synthesis were obtained. These functions were 2.0-3.3-fold higher than those obtained by the same period of monolayer culture. In addition, final attained unit cell-based functions on day 15 were almost comparable to the levels reported for cultures of adult porcine hepatocytes in both monolayer and 3D spheroid cultures. These results demonstrate that the use of a biodegradable polymer-based 3D culture with an appropriate combination of biofactors is a promising approach to maximize functional maturation of hepatocyte progenitors from large animals. In addition, the established culture conditions are worth using to engineer large liver tissue equivalents for pigs in large-animal-based preclinical studies.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Cell surface molecules are not only important for cell-cell interactions but also useful for a marker to define cell types and differentiation stages. Unlike hematopoietic system in which numerous such antigens have been identified, only a few cell surface molecules have been used to define differentiation stage of hepatocytes. In order to identify such cell surface molecules, we performed DNA microarray analysis using mRNA from fetal hepatocytes in E12.5 and E17.5 mice and cDNAs encoding a membrane protein were selected. Northern blot analysis was employed to confirm the genes upregulated during maturation of fetal hepatocytes and neuritin, a GPI-anchored protein, was found as a membrane protein expressed in hepatocytes, but not in nonparenchymal cells. Its expression increased along with liver development and the maximum expression was achieved from the neonatal to adult stage. The neuritin protein was localized in sinusoidal lumen of hepatocytes in adult liver. Partial hepatectomy transiently downregulated the expression of neuritin. The expression of neuritin mRNA in C/FBP alpha deficient liver was reduced to about 50% of that of wild type mice. Thus, neuritin expression is well correlated to the maturation of hepatocytes and can be a useful tool to define the differentiation stage of hepatocytes. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.07.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

To assess the feasibility of liver tissue equivalents based on selective propagation and differentiation of hepatocyte progenitors in three-dimensional (3D) culture, the efficacy of fetal mouse liver cells cultured in poly-L-lactic acid ( PLLA) scaffolds in the presence of nicotinamide, dimethyl sulfoxide, and oncostatin M was investigated both in vitro and in vivo. The albumin production of PLLA-cultured fetal mouse liver cells in the presence of these three factors was remarkably enhanced with culture time, and after 4 weeks it attained almost the same production found in adult mouse hepatocytes cultured for 3 days in PLLA scaffolds, based on the unit DNA amount. In addition, implantation of engineered liver tissue based on this in vitro PLLA culture system into the peritoneal cavity of 70% hepatectomized mice showed a remarkably higher presence of albumin-positive engrafted cells 15 days after the operation when compared with fetal mouse liver cells or adult mouse hepatocytes freshly isolated and cultured for 1 day. These results demonstrate that the basic concept regarding the engineering of liver tissue equivalents based on in vitro selective propagation and differentiation of hepatocyte progenitors in 3D biodegradable scaffolds shows promise for future liver tissue engineering.

DOI： 10.1089/ten.2004.10.1577

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To organize a liver tissue of a clinically meaningful size, in vitro large-scale control of growth and maturation of fetal liver cells in biodegradable polymer scaffolds is necessary. Therefore, the supporting function for fetal liver cells and the effects of oncostatin M (OSM), nicotinamide (NA) and dimethyl sulfoxide (DMSO) on growth and hepatic differentiation in poly-L-lactic acid (PLLA) scaffolds were studied in vitro. After preparing three-dimensional (3D) PLLA scaffolds having a well-developed open-pore structure using NH4HCO3 particles, fetal liver cells obtained from E14.5-C57BL/6CrSlc-murine embryos were inoculated in the scaffolds. Cells were cultured in Williams' E medium with or without OSM, NA and DMSO for 4 weeks with a medium change every 2 days. Results showed that numerous cells spread over the surface of the PLLA and a large amount of extracellular matrix in PLLA cultured fetal liver cells with NA/DMSO/OSM group compare with basal group. In addition, significant increases in cell number and albumin secretion were also observed in NA/DMSO/OSM group than in basal group. These results show that OSM, NA and DMSO remarkably stimulated proliferation and maturation of hepatic parenchymal cells in vitro in terms of morphology and liver-specific functions. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mscc.2003.12.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WICHTIG EDITORE  

Fetal human liver cell fractions, which contain large numbers of hepatocyte progenitors, have high proliferation potential in vitro. To create an engineered liver tissue equivalent of a clinically significant size, however, repeated subcultivation and functional maturation are necessary in vitro. A commercially available human fetal liver cell fraction that was cultivated for some time in vitro has been reported to lose liver specific functions almost completely. We therefore investigated the effects of oncostatin M (OSM) and hepatocyte growth factor (HGF) in long term three-dimensional (3D) culture using macroporous poly-L-lactic acid (PLLA) scaffolds on the restoration of such liver-specific functions of the fraction.
3D culture using PLLA scaffolds with OSM remarkably enhanced the albumin production and cytochrome P450 1A1/2 capacity with the culture time. HGF alone had no preferable effect on these functions even in 3D culture. alpha-fetoprotein production was consistently suppressed in the 3D culture compared with that in monolayers. This suppression was not observed in the same types of culture of hepatocarcinoma Hep G2 cells.
Despite these favorable observations on the 3D culture with OSM, the final attained functional levels at the 5th week were still over ten-times lower than those of Hep G2 cells when standardized with a cellular DNA amount. Although further improvement is needed for the complete functional restoration and maturation in vitro, these results demonstrate that a combination of 3D culture using PLLA scaffolds and OSM offers promising culture conditions for in vitro maturation of human hepatocyte progenitors.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO  

Previously, we described that embryonic day 14.5 (E14.5) mouse fetal hepatocytes differentiate to express tyrosine amino transferase (TAT) and glucose-6-phosphatase, which are expressed in the perinatal liver, in response to oncostatin M (OSM) or in high-cell-density culture. However, under such conditions, fetal hepatic cells failed to express genes for adult liver-specific enzymes, such as tryptophan oxygenase (TO). Although phenobarbital (PB) and dimethylsulfoxide (DMSO) have been known to maintain the functions of adult hepatocytes in vitro, they failed to induce TO expression in fetal hepatic cells. Thus far, no system has been developed that reproduces terminal differentiation of fetal hepatocytes in vitro. Here, we describe that extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma (EHS) in combination with OSM or high-cell-density culture induced expression of TO as well as cytochrome P450 genes that are involved in detoxification. However, EHS alone was insufficient to induce expression of TO, although it induced TAT expression in fetal hepatocytes. In addition, high-density culture further augmented differentiation. In conclusion, the combination of signals by cytokines, cell-cell contact, and cell-matrix interaction is required for induction of adult liver functions in fetal hepatocytes in vitro. This primary culture system will be useful for studying the mechanism of liver development.

DOI： 10.1053/jhep.2002.33331

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COGNIZANT COMMUNICATION CORP  

To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly-L-lactic acid (PLLA) for fetal liver cells and the effects of oncostatin M (OSM) on hepatic differentiation were studied. After preparing three-dimensional biodegradable PLLA scaffold having a well-developed open-pore structure by a gas-forming method with ammonium chloride particles as a porogen and a gas-forming reagent, fetal liver cells separated from E14.5-C57BL/6CrS1c murine embryos were inoculated in the PLLA scaffolds. Cells were cultured in Williams' E medium with or without OSM (10 ng/ml) for 30 days with a medium change every 2 days. Results showed that there were significant increases in the number of cells and in albumin secretion in PLLA culture compared with in monolayer culture on day 15. In addition, a significant increase in albumin secretion was observed in OSM-added PLLA culture compared with OSM-free culture, and there was only a slightly enhanced albumin secretion in monolayer cultures with OSM. These results suggest that PLLA may enhance the biological activity of OSM for inducing maturation of fetal liver cells. Interestingly, the number of cells in PLLA culture with OSM decreased compared with OSM-free PLLA culture at day 15. This may be because promotion of hepatic development by OSM simultaneously suppressed in vitro hematopoiesis (i.e., blood cell production). In summary, our results indicate that the three-dimensional PLLA scaffold is a good support material for the cultivation of fetal liver cells and that OSM is capable of not only terminating hematopoiesis of the fetal liver but also stimulating the maturation of hepatic parenchymal cells in vitro.

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Although cells isolated from fetal liver are one of the major sources for liver tissue engineering, it is still very difficult to induce them to fully differentiate in vitro into mature hepatocytes. We therefore investigated the effects of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) on differentiation in terms of the expression of various liver-specific functions, because these factors have been reported to induce the emergence of possible hepatocyte progenitor cells (small hepatocytes) in adult rat hepatocyte culture or maturation of fetal mouse liver cells in culture. Fetal liver cells isolated from mouse embryos were cultured for 5 weeks in collagen-precoated plates. NA (10 mM) and DMSO (1%) remarkably enhanced the emergence of small hepatocytes, and OSM also synergistically enhanced the selective growth of small hepatocytes and inhibited the growth of blood cell populations. In the presence of these three factors, such small hepatocytes became dominant in culture, so that they covered almost 60-70% of confluence after week 2. In addition, some of them piled up over the small hepatocyte monolayer and displayed distinctively differentiated morphology, such as the emergence of binucleated cells, formation of tight gap junctions, and possible bile duct structures. Although OSM alone had very weak effects on hepatocyte functions, albumin secretion and cytochrome P450IA1/2 capacity were greatly enhanced when combined with NA or DMSO. This functional observation closely agreed with the emergence of small hepatocytes. In contrast, ammonium removal was strongly dependent on DMSO alone. DNA amount basis functions of fetal cells with three factors at week 5 were 1/7 for albumin secretion, 3 times higher for ammonium removal, and 1/10 for P450 capacity, compared with those of cultured adult mouse hepatocytes. These results show that inclusion of NA, DMSO, and OSM in the culture medium significantly enhances in vitro maturation of fetal liver cells when compared with conventional culture conditions.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

We previously demonstrated that oncostatin M (OSM) promotes hepatic development in concert with glucocorticoid. The livers from mice deficient for gp130, a signaling subunit of the OSM receptor, displayed reduced expression of hepatic differentiation marker and defective glycogenic function. However, these phenotypes were not completely abolished in gp130(-/-) mice, suggesting that there is an alternative pathway regulating hepatic development in vivo. To test this possibility, we cultured gp130(-/-) fetal hepatic cells and investigated a signal that induces hepatic differentiation. When hepatocytes were forced to interact with each other by inoculating cells at high densities, hepatic differentiation was induced even in the absence of gp130. Moreover, cells stimulated with OSM and/or cultured at a high density possess many other metabolic functions. These observations suggest that fetal hepatic cells acquire multiple characteristics of differentiated hepatocytes in response to the signals generated by cell-cell contacts as well as by OSM. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3635

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line, Huh 7, at different concentrations of glutamine, glucose and ammonia was examined. During the incubation of the primary hepatocyte cells, glutamine and ammonia concentrations decreased, that of urea increased, and that of glucose remained the same. In the case of Huh 7 cells, glucose was consumed rapidly, the concentration of ammonia increased and that of urea remained the same. The major energy sources among medium components were glutamine for the primary cells and glucose for Huh 7 cells, although the primary hepatocytes may utilize intracellular glycogen as energy source. As the glutamine concentration in the incubation medium increased, the specific rates of not only glutamine consumption, but also ammonia production by the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primary cells increased then. Increase of glucose concentration had no effect on glutamine and ammonia metabolism by both cells, although it increased glucose consumption by Huh 7 cells. The incubation of the primary cells with higher ammonia concentration increased all specific rates of glutamine consumption, ammonia consumption and urea production. An increase in the ammonia concentration to 5 mM changed the ammonia metabolism from production to consumption and increased the specific glucose consumption rate. Consequently, increases in the glutamine and ammonia concentrations were revealed to have negative and positive effects, respectively, on decreasing ammonia concentration by both of rat primary hepatocytes and Huh 7 cells.

DOI： 10.1023/A:1008165027319

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Language：Japanese   Publisher：日本電気泳動学会  

がん微小環境には細胞外マトリックス(ECM;Extracellular Matrix)が含まれており,ECMはがん細胞アッセイを行う際に用いられている.しかし現行のアッセイ系ではコラーゲンなど単一もしくは特定のECM成分しか用いられておらず,がん細胞の行動特性を評価するアッセイとしては改良の余地がある.細胞の機能を調べるための新しいプラットフォームとして近年注目されている脱細胞化組織をゲル化させ細胞培養に利用することで,よりin vivoに近い条件でがん細胞のアッセイを実施可能であると考えた.脱細胞化ゲル(decellularized tissue gels;DTGs)ががん細胞に与える影響を調べるために,DTGsをコーティングしたプレート上でがん細胞株を培養した.また,DTGsをSDS-PAGEにより分離後,銀染色を行うことで含有タンパク質を調べた.結果,DTGsにはがん細胞の増殖をin vitroで変動させる効果があることが判明した.銀染色では複数のタンパク質バンドが出現したため,DTGsには単一の成分のみではなく,in vivoのように複数の成分からなるECMの含有が期待できる.以上より,DTGsはがん細胞のin vitroアッセイに有用なツールとして検討する価値があるといえる.(著者抄録)

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Multicellular spheroid (MCS) culture has been believed to mimic three-dimensional environments in our body. For instance, hepatocytes show higher functions in the MCS condition. The inner structure of the MCS is, however, not well organized, and engineering of the configuration would be effective to represent further liver characteristics. This chapter will show how to reconstruct the liver-like tissues in vitro by the engineering of MCS those are based on a swelling phenomenon of a culture medium dissolving polymer molecule. The unique method enables us to engineer the microarchitectures in the MCS by formation of void spaces or filling of extracellular matrices. The liver functions/features are enhanced/improved by regulating microenvironment. Fabrication of the MCS with microstructures would achieve a breakthrough in study for stem cells and cancer, animal testing alternative, human-on-a-chip, and regenerative medicine.

DOI： 10.1016/B978-0-12-812301-0.00008-6

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The pancreatic islet is a functional unit of the pancreatic endocrine system, and it has a potential to cure type 1 diabetes by transplantation. However, donor shortage is a serious problem. To solve this, it is suggested to use the islet-like tissues from pancreatic beta cell lines or iPS cell derived insulin-producing cells. In this paper, we introduce a three-dimensional culture method for constructing islet-like tissues with microstructures those can enhance the insulin secretion activity. The islet-like tissues showed higher insulin secretion activity by mixing α cells and/or hydrogel beads. Moreover, the islet-like tissues containing pancreatic alpha cells exhibited higher therapeutic performance when they were transplanted under the kidney capsules of the streptozotocin-induced diabetic mice. Achievement to develop more functional islet-like tissues results in a reduction of medical cost as well as cell number in transplantation. The concept of redesigning the microstructure of islet-like tissues may be effective for future transplantation treatment.

DOI： 10.11378/organbio.24.213

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To elucidate the mechanism of hematopoiesis and bone marrow (BM) diseases, the realization of BM three-dimensional (3D) culture system is very important. Because more than 90% of the BM cells are non-adherent blood cells, it is difficult to organize the dispersed-state BM cells with the conventional aggregation methods e.g. hanging drop method. The objective of this study is to reproduce the BM-like tissue from dispersed-state BM cells in vitro. We reported a method of forming aggregates using methylcellulose (MC) medium [1]. This method can make a 3D aggregation state rapidly regardless of adhesive property of the cells. This method applied to the BM cells, they gathered in 10 minutes and formed tissue-like spheroids within 24h. Formed BM-like tissue could remove from MC medium on the Day 1. By hematoxylin-eosin staining, the blood cells were maintained for 3 days. By staining with anti-CD68 antibody, macrophages in the BM-like tissue were found at the same frequency as those in the intact BM tissue. It was confirmed that mesenchymal cells, for example CXCL12+ reticular cells, PDGFR and alpha
+ cells were maintained in the BM-like tissues. From these results, the method using MC medium was suitable to reconstitute the BM-like tissue.

DOI： 10.1109/MHS.2015.7438268

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Language：English   Publisher：Institute of Electrical and Electronics Engineers Inc.  

A typical three-dimensional culture of hepatocytes is to embed cells in extracellular matrix (ECM) capsule by using an emulsion method. However, usage of oil causes cellular damage. In this study, we aimed to establish a new method for forming cell-embedded ECM capsules without using oil. We also report ECM-loaded spheroids which is a different type of cell-embedded ECM capsules having less amount of ECM. To form ECM capsule, we utilized the methylcellulose (MC) medium [1]. We injected 1 μl of undiluted ECM (Matrigel or collagen) suspending 2000 Hep G2 cells into the MC medium. The cellular damage was obviously reduced when we made capsules by the MC medium. However, the albumin secretion activity of the Hep G2 cells in ECM capsule was half level compared with normal multicellular spheroids because too much gels inhibited cell-to-cell interaction. When we used 30-times diluted Matrigel, the thickness of gel between cells was significantly reduced. In this study, we called this kind of spheroid with ECM as ECM-loaded spheroid. The albumin secretion activity of ECM-loaded spheroids was higher than that of normal spheroids. In conclusion, we established a new method to fabricate ECM capsules and ECM-loaded spheroids, and ECM-loaded spheroids demonstrated higher hepatic function than conventional spheroids.

DOI： 10.1109/MHS.2015.7438332

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Language：English   Publisher：IEEE  

Compared with monolayer cultures, three-dimensional cultures such as multicellular spheroids provide the high cellular functions and the in vivo-like microstructures. Extracellular matrix (ECM) is one of the most important regulators of in vivo tissues' functions and microstructures. A typical three-dimensional culture for assembling spheroid and ECM is to embed spheroid in ECM gel. However, the effect of ECM on the inner of spheroid was not observed because spheroid is embedded in ECM after fabricating spheroid. In the last symposium on MHS, we reported a new method for fabricating ECM containing spheroid which the ECM layer exists between cells in spheroid. In this study, we aimed to reveal whether ECM is able to regulate functions and microstructures of hepatic ECM containing spheroid. To fabricate ECM containing spheroid, we utilized the 3% methylcellulose (MC) medium [1]. We injected 1 mu l of diluted ECM solution (Matrigel or collagen) suspending 2000 of Hep G2 cells into the MC medium. To examine contribution of ECM to spheroids, the albumin secretion was measured. The albumin secretion activity of Matrigel containing spheroids was higher than that of normal spheroids. By contrast, when we used type I collagen, the activity was lower than that of normal spheroids. However, type I collagen enhanced cellular proliferation of Hep G2 cells. In addition, we found that Matrigel containing spheroids was effective to form bile canaliculi in spheroids. These results indicated that ECM was able to regulate functions and microstructures of hepatic multicellular spheroids.

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The objective of this study was to establish a method enabling us to control microarchitectures of spheroids. We injected 1 μl of culture medium suspending Hep G2 cell and alginate hydrogel beads into a 3% methylcellulose medium. The beads were 20 μm in diameter. The injected cells and beads were rapidly aggregated in 10 min, and formed hybrid spheroids in 24 h. Microchannel structures were constructed by enzymatic digestion of the beads. In a different experiment, injection of Hep G2 cells and HUVECs resulted in forming microstructures by autonomous cellular rearrangement. Another type of micropattern was able to be obtained by injecting HUVECs and small-spheroids. Dissolved polymeric molecules like ECMs were also possible to be concentrated together with Hep G2 cells. The amount of polymeric molecules existing inside of the spheroids was tunable by changing its concentration. These data show that our method is useful to engineer microarchitectures of spheroids.

DOI： 10.11239/jsmbe.54annual.s348

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Evaluation of biological activity of multicellular spheroids with label-free technique is quite important. Therefore, we focused on mechanical characterization of spheroids using force sensor probe using quartz crystal resonator (QCR). In this study, we measured the mechanical characteristics of HepG2 spheroid with different culture periods.

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Language：Japanese   Publisher：日本組織培養学会  

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Language：English   Publisher：IEEE  

The goal of this article is to regulate the structure of multicellular spheroids. Some epithelial cells including fetal hepatocytes have tendency to form "cyst" structure when they cultured in 3 dimensional aggregates. We added hydrogel beads to the aggregates and obtained "densely-packed" structure with microchannels enabling gas/nutrients exchange. In addition of the structural change, albumin secretion, a typical function of hepatocytes, was also improved. This technique helps to maintain the living cells with functions in multicellular spheroids.

DOI： 10.1109/MHS.2014.7006165

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Language：English   Publisher：Springer-Verlag London Ltd  

In this chapter, we introduce and summarize the results from our groups on typical macroporous and biodegradable poly-L-lactic acid (PLLA) scaffolds-based 3D shaking culture of fetal hepatocyte populations isolated from mice, rats, and pigs, and on some preliminary implantation of the cell-loaded scaffolds to mice and rats. In such 3D microenvironment, inoculated cells were organized into heterogenic 3D aggregates or multilayers, functional levels, and their in vitro stability was greatly enhanced when compared with those in 2D monolayer cultures. Although detoxification capacity in terms of EROD measurement did not seemed to be fully matured, other typical functions such as albumin production attained the adult level. This was enabled by the synergistic effects of 3D culture and soluble factors cocktails. Combination of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) was very effective in fetal mice culture, but it does not support the growth and maturation of fetal rat hepatocytes, for which other cocktail composed of NA, HGF, FGF-1, FGF-4, OSM, and sodium butyrate was effective. In the case of fetal porcine hepatocytes, presumably because the obtained hepatocytes were in better matured stage than mice and rats, dependency on soluble factors was low, and 3D culture itself remarkably enhanced their spontaneous growth and maturation. The biggest problem in such 3D culture, cellular growth was limited only to the periphery of macropores of the scaffolds even with the thin disk shape of the scaffolds and with continuous shaking, resulted in about at most several times growth and one-tenth cellular density that in vivo. This indicated the insufficient mass transfer (primarily oxygen) between culture medium and inner spaces of the scaffolds. However, upon implantation to mesentery leaves of animals, almost all the remaining spaces in the scaffolds were finally filled with proliferated hepatocytes in mice and rats. These results clearly demonstrate that fetal cells that grow and mature in 3D culture with appropriate cocktails of soluble factors show promise in partly supporting the insufficient host liver functionality upon implantation.

DOI： 10.1007/978-1-4471-4171-6_4

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Language：Japanese   Publisher：Institute of Industrial Science The University of Tokyo  

In this study, we established a mathematical model of the self-organization of two types of the cells, which depends on cell-to-cell interactions. To observe the behaviors of the cells, we used polydimethylpolysiloxane (PDMS) microdevice chambers to enhance cell migration by supplying O2. Based on time-lapse observations, we hypothesized three simple rules for self-organization and produced a mathematical model to emulate cell behavior. We confirmed that the self-organized patterns produced by the cell culture and the computer simulation were not significantly different. These findings may be useful for understanding the regeneration of tissues/organs in vitro.

DOI： 10.11188/seisankenkyu.65.337

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

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This paper reports a method to fabricate liver tissue spheroids with microchannel network in which 1 !m diameter particles can pass through. We made alginate hydrogel beads that have 20 !m in diameter, and formed heterogeneous spheroids consisting of about 1,000 of hepatic cells and same number of the beads. One day culture of the hetero-spheroids resulted in network-like organization of the hepatic cells. The alginate beads were then digested by alginate lyase treatment for 10 min. The connectivity of porous spheroids was confirmed using 1 !m fluorescence beads or a micro device. These data suggest that bottom-up approach to make microchannel is effective in engineering of multi-cellular spheroids.

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In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues
cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-l-lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D microfabrication processes, final cell densities were less than one-tenth of those of in vivo liver tissues
cells preferentially grew along the surfaces of the channels and this fact suggested the necessity of improved 3D fabrication technologies with higher resolution. In any case, suitable oxygen supply, meeting the cellular demand at physiological concentrations, was the most important factor that should be considered in engineering liver tissues. This enables cells to utilize aerobic respiration that produces almost 20 times more ATP from the same glucose consumption than anaerobic respiration (glycolysis). This also allows the cells to exhibit their maximum reorganization capability that cannot be observed in conventional anaerobic conditions. © 2012 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-61779-468-1_16

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Language：Japanese   Publisher：東京大学生産技術研究所  

A method for direct cell-to-cell binding has high potential to make reconstituted tissues and organs for pharmacological applications and regenerative medicine. It may be possible to produce unique materials and devices, which are based on biological properties by arranging cells appropriately. In addition, an ambitious study might lead to the construction of "multicellular artificial life" by assembling single cells. In this report we show a technology to attach cells to each other rapidly to make heterogeneous multicellular spheroids and minutely arranged cell-based micro tissues.

DOI： 10.11188/seisankenkyu.63.333

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In this report, we demonstrate a new approach to assemble micro-tissues with precise cell positioning based on the integration of a powerful cell-to-cell adherence system, avidin-biotin binding system (ABBS), optical tweezers and microdevices. Interestingly, avidinylated and biotinylated cell adherence occurred within 1 second using laser trapping, enabling single cell manipulation. We showed precise, direct single-cell-based tissue assembly using ABBS, optical tweezers and microdevices, followed by damage-free tissue culture. This approach to make micro-tissue has considerable potential for use in application such as tissue engineering, regenerative medicine, and drug screening system.

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Improvement of primary culture of hepatocytes is definitely important for in vitro toxicity tests for various chemicals. Various improvements in terms of culture system have been investigated, such as enhancement in oxygen supply to the cells, coculture with nonparenchymal cells or cell lines, 3D organization into spheroids, etc. However, it is still difficult to maintain in vivo-like high liver-specific functions in in vitro culture. In this report, we briefly explained both the related important research and latest new approach of our research that tried to mimic the in vivo diurnal ch...

DOI： 10.11188/seisankenkyu.60.152

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Language：Japanese   Publisher：The University of Tokyo  

&nbsp;&nbsp;&nbsp;Improvement of primary culture of hepatocytes is definitely important for <i>in vitro</i> toxicity tests for various chemicals. Various improvements in terms of culture system have been investigated, such as enhancement in oxygen supply to the cells, coculture with nonparenchymal cells or cell lines, 3D organization into spheroids, etc. However, it is still difficult to maintain in vivo-like high liver-specific functions in <i>in vitro</i> culture. In this report, we briefly explained both the related important research and latest new approach of our research that tried to mimic the in vivo diurnal changes of hepatic functions.

DOI： 10.11188/seisankenkyu.60.152

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DOI： 10.11491/scej.2007.0.385.0

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DOI： 10.11491/scej.2007.0.381.0

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DOI： 10.11491/scej.2007.0.380.0

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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We have been investigating the feasibility of bottom-up tissue engineering, a new methodology to form tissues, by assembling single cells one by one using an avidin-biotin binding system (ABBS). In this report, we used optical tweezers in addition to ABBS to form small tissues where the cells alignment was precisely controlled. ABBS enabled very quick attachment of human hepatoma Hep G2 cells, and single cell manipulation by optical tweezers aligned the cells accurately. These results show that the combination of two technologies, ABBS and single cell manipulation, provides a new methodology for bottom-up tissue engineering.

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For engineering implantable liver tissues, we designed a new scaffold with a three-dimensional (3D) branching and joining flow channel network (inner diameter is 1 mm) described by accumulated unit tetrahedrons (edge length is 4 mm). For its fabrication, biodegradable polycaprolactone (PCL) and 80% (w/w) of NaCl salt particles as a porogen were completely mixed and applied to the selective laser sintering (SLS) process, a rapid prototyping technique. We successfully obtained a scaffold that had a high porosity of 89% with a pore size of 100-200 μm and a 3D network flow channels whose inner diameter is 800 μm. Results of X ray CT confirmed the sintered scaffolds had completely interconnected flow channels as designed. A preliminary perfusion culture over 9 days demonstrated that such mirochannels was necessary to guide the cells to grow and function.

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

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Language：Japanese   Publisher：豊田理化学研究所  

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DOI： 10.11188/seisankenkyu.57.87

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DOI： 10.11188/seisankenkyu.57.87

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Publisher：The Society of Life Support Technology  

DOI： 10.5136/lifesupport.17.Supplement_101

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DOI： 10.5136/lifesupport.17.Supplement_101

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DOI： 10.5136/lifesupport.17.Supplement_100

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DOI： 10.5136/lifesupport.17.Supplement_100

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Language：Japanese   Publisher：日本再生歯科医学会  

There are still two major problems that have not been overcome in artificial liver support systems, although they have been studied for long years. One of the problems is about the source of cells, which can proliferate and differentiate as we desired. The other is construction of blood vessels or micro-channels to exchange oxygen and nutrients. Recently, proliferative fetal hepatocytes are gradually recognized as remarkable cell source, because energetic studies in the field of molecular biology have enabled differentiation of proliferative fetal hepatocytes in vitro. In addition, transpla...

DOI： 10.11223/jard.2.93

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Language：Japanese   Publisher：一般社団法人 日本人工臓器学会  

DOI： 10.11392/jsao1972.33.2supplement_s133

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：COGNIZANT COMMUNICATION CORP  

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Language：Japanese   Publisher：(一財)バイオインダストリー協会  

肝臓は重要な代謝器官であるが,殆どの代謝機能は,出生前後において急速に獲得される.著者等は,この劇的な変化のメカニズムを分子生物学の手法を用いて明らかにした.著者等が行っている肝発生研究の成果を医療分野へと応用する可能性について述べた

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Other Link： http://search.jamas.or.jp/link/ui/1998005273

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Applicant：財団法人神奈川科学技術アカデミー

Application no：特願2003-393848  Date applied：2003.11

Announcement no：特開2005-156295  Date announced：2005.6

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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Award type：Award from international society, conference, symposium, etc.  Country：Japan

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

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