Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

自動培養装置によるヒトiPS細胞の長期間培養に成功 . 京都大学プレスリリース. 2015-11-27.Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells

High-throughput analyses of gene functions on a cell chip by electroporation.

Fig.1は次の論文のFig.1を引用 Fujimoto, H., Kato, K., Iwata, H. (2008) Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Anal. Bioanal. Chem. 392, 1309–1316.Fig.2は次の論文のFigure 7を引用 Yamauchi, F., Kato, K., Iwata, H. (2004) Spatially and temporally controlled electroporation of adherent cells on plasmid DNA-loaded electrode. Nucleic Acids Res. 32, e187.Fig.3は次の論文のFig.6を引用 Fujimoto, H., Kato, K., Iwata, H. (2008) Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Anal. Bioanal. Chem. 392, 1309–1316Genome-wide functional annotation of genes is one of the major challenges in current biology. Such investigation requires a high-throughput methodology for efficient and parallel overexpression or silencing of multiplexed genes in living cells. The transfection method described here employs an electric pulse and a cell-chip technology, and provides the possibility of analyzing gene functions in a high-throughput manner

Surface modification of islets with PEG-lipid for improvement of graft survival in intraportal transplantation.

BACKGROUND: Transplantation of islets of Langerhans (islets) is a promising technique for treating insulin-dependent diabetes mellitus (type I). One unsolved issue is the early graft loss due to inflammatory reactions triggered by blood coagulation and complement activation that occurs immediately after transplantation into the liver through the portal vein. Several proposed approaches for improvement of the graft survival include heparin coating and covalent poly(ethylene glycol) (PEG) conjugation. We previously have studied the improvement of graft survival by modification of islet surfaces using amphiphilic PEG-conjugated phospholipid and bioactive molecules. Here, we analyzed the effect of PEG-modification on the improvement of graft survival immediately after intraportal transplantation into streptozotocin-induced diabetic mice. METHODS: The surface of hamster islets was modified with PEG-lipid. PEG-lipid modified islets (PEG-islets) were transplanted into the liver through the portal vein of streptozotocin-induced diabetic mice. We measured the graft survival periods and blood insulin levels immediately after intraportal transplantation to determine the cell damage to islets. Histocytochemical analyses of liver were also performed postintraportal transplantation. RESULTS: The graft survival of PEG-islets was significantly prolonged compared with bare islets in livers of diabetic mice. Reduction of blood insulin level within 60 min after transplantation of PEG-islets suggests that the cell damage observed immediately after transplantation could be suppressed by surface modification with PEG in comparison with bare islets. CONCLUSION: Our approach for the improvement of graft survival will be useful in the clinical setting

Cell surface modification with polymers for biomedical studies

Surface modification of living cells with natural or synthetic polymers is a powerful and useful tool in biomedical science and engineering. Various functional groups and bioactive substances can be immobilized to the cell surface through covalent conjugation, hydrophobic interaction, or electrostatic interaction. In this review, we provide an overview of the methods and polymers employed in cell surface modification, including: (1) covalent conjugation utilizing amino groups of cell surface proteins, (2) hydrophobic interaction of amphiphilic polymers with a lipid bilayer membrane, and (3) electrostatic interactions between cationic polymers and a negatively charged cell surface. We also discuss their applications in studies on cell therapy, cell–cell interaction analysis, cell arrangement, and lineage determination of stem cells

ハンノウセイ コウブンシ ノ ゴウセイ ト コウブンシ - コウブンシカン ハンノウ ニ カンスル ケンキュウ

京都大学0048新制・課程博士工学博士甲第2296号工博第636号新制||工||455(附属図書館)UT51-54-X32京都大学大学院工学研究科高分子化学専攻(主査)教授 北丸 竜三, 教授 中島 章夫, 教授 今西 幸男学位規則第5条第1項該当Kyoto UniversityDFA

Surface plasmon resonance in monitoring of complement activation on biomaterials.

When artificial materials come into contact with blood, various biological responses are induced. For successful development of biomaterials used in biomedical devices that will be exposed to blood, understanding and control of these interactions are essential. Surface plasmon resonance (SPR) spectroscopy is one of the surface-sensitive optical methods to monitor biological interactions. SPR enables real-time and in situ analysis of interfacial events associated with biomaterials research. In this review, we describe an SPR biosensor and its application to monitor complement activation onto biomaterials surface. We also discuss the effect of surface properties of the material on complement activation

Complement activation on degraded polyethylene glycol-covered surface

Surface modification with polyethylene glycol (PEG) has been employed in the development of biomaterials to reduce unfavorable reactions. However, unanticipated body reactions have been reported, with activation of the complement system being suggested as having involvement in these responses. In this study, we prepared a PEG-modified surface on a gold surface using a monolayer of alpha-mercaptoethyl-omega-methoxy-polyoxyethylene. We observed neither protein adsorption nor activation of the complement system on the PEG-modified surface just after preparation. Storage of the PEG-modified surface in a desiccator under ambient light for several days or following ultraviolet irradiation, reflection-adsorption (FTIR-RAS) and X-ray photo spectrometry revealed deterioration of the PEG layer, which became a strong activator of the complement system through the alternative pathway

Layer-by-layer co-immobilization of soluble complement receptor 1 and heparin on islets.

Early graft loss due to instant blood-mediated inflammatory reactions (IBMIRs) is a major obstacle of clinical islet transplantation; inhibition of blood coagulation and complement activation is necessary to inhibit IBMIRs. Here, human soluble form complement receptor 1 (sCR1) and heparin were co-immobilized onto the surfaces of islet cells. sCR1 molecules carrying thiol groups were immobilized through maleimide-poly(ethylene glycol)-phospholipids anchored in the lipid bilayers of islet cells. Heparin was immobilized on the sCR1 layer via the affinity between sCR1 and heparin, and additional layers of sCR1 and heparin were formed layer-by-layer. The sCR1 and heparin molecules in these layers maintained anti-complement activation and anti-coagulation activities, respectively. This promising method could be employed to reduce the number of islet cells required to reverse hyperglycemia and prolong graft survival in both allo- and xeno-islet transplantation

Immobilization of the soluble domain of human complement receptor 1 on agarose-encapsulated islets for the prevention of complement activation.

The transplantation of islets of Langerhans has been successfully applied to the treatment of insulin-dependent diabetes. However, a shortage of human donors is the hardest obstacle to overcome. We aimed to develop a bioartificial pancreas that can realize xeno-islet transplantation. The islets were encapsulated in agarose microbeads carrying the soluble domain of human complement receptor 1 (sCR1), which is an effective inhibitor of the classical and alternative complement activation pathways. When naked rat islets were cultured in rabbit serum, large amounts of insulin leaked from the damaged islets over the course of a few days incubation, but no damaged cells were observed among islets in sCR1-agarose microbeads cultured in rabbit serum for 4 days. Although low levels of insulin were detected in the rabbit serum, the insulin did not leak from damaged β-cells, it was physiological insulin secreted by the β-cells