Pub Date : 2015-08-26DOI: 10.3727/215517915X689074
Sreenadh Sasidharan Pillai, H. Yukawa, D. Onoshima, V. Biju, Y. Baba
Quantum dots (QDs) have recently been investigated as fluorescent probes for detecting a very small number of biomolecules and live cells; however, the establishment of molecular imaging technology with on-off control of QD fluorescence remains to be established. Here we have achieved the fluorescence off state of QDs with the conjugation of black hole quencher (BHQ) molecules intermediated with peptide by using streptavidin-QDs585 and biotin-pep-BHQ-1. The fluorescence of streptavidin-QDs585 was decreased by the addition of biotin-pep-BHQ-1 in a dose-dependent manner. It has been suggested that the decrease in QDs585 fluorescence occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of fluorescence intensity and lifetime of streptavidin-QDs585 and QDs585-pep-BHQ-1. QDs585 fluorescence could be quenched by more than 60% efficiency in this system. The sequence of intermediate peptide (pep) was GPLGVRGK, which can be cleaved by matrix metalloproteinases (MMPs) produced by cancer cells. QDs585-pep-BHQ-1 is thus expected to detect the MMP production by the recovery of QDs585 fluorescence as a new bioanalytical agent for molecular imaging.
{"title":"Fluorescence Quenching of CdSe/ZnS Quantum Dots by Using Black Hole Quencher Molecules Intermediated With Peptide for Biosensing Application.","authors":"Sreenadh Sasidharan Pillai, H. Yukawa, D. Onoshima, V. Biju, Y. Baba","doi":"10.3727/215517915X689074","DOIUrl":"https://doi.org/10.3727/215517915X689074","url":null,"abstract":"Quantum dots (QDs) have recently been investigated as fluorescent probes for detecting a very small number of biomolecules and live cells; however, the establishment of molecular imaging technology with on-off control of QD fluorescence remains to be established. Here we have achieved the fluorescence off state of QDs with the conjugation of black hole quencher (BHQ) molecules intermediated with peptide by using streptavidin-QDs585 and biotin-pep-BHQ-1. The fluorescence of streptavidin-QDs585 was decreased by the addition of biotin-pep-BHQ-1 in a dose-dependent manner. It has been suggested that the decrease in QDs585 fluorescence occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of fluorescence intensity and lifetime of streptavidin-QDs585 and QDs585-pep-BHQ-1. QDs585 fluorescence could be quenched by more than 60% efficiency in this system. The sequence of intermediate peptide (pep) was GPLGVRGK, which can be cleaved by matrix metalloproteinases (MMPs) produced by cancer cells. QDs585-pep-BHQ-1 is thus expected to detect the MMP production by the recovery of QDs585 fluorescence as a new bioanalytical agent for molecular imaging.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"8 1-2 1","pages":"57-62"},"PeriodicalIF":0.0,"publicationDate":"2015-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517915X689074","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-08-26DOI: 10.3727/215517915X689056
Y. Miyamoto, M. Ikeuchi, H. Noguchi, T. Yagi, S. Hayashi
In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 µm × 500 µm) and a bottom aperture (300 µm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.
在药物发现中，对生物体内的肝细胞进行评估是非常重要的。与2D培养方法相比，肝细胞3D培养技术的发展已经成功地维持了长期的肝脏功能，形成了肝脏特异性结构。进行药物检测的关键是建立稳定的体外评价体系。在这篇文章中，我们报道了一种用于体外培养肝球体的锥形支架(TASCL)装置。TASCL装置将作为药物发现的毒性评价系统。TASCL装置的总尺寸为10 mm × 10 mm，包含400个微孔，每个微孔的顶孔(500µm × 500µm)和底孔(直径300µm的圆形)。我们评估了TASCL装置中HepG2球体的形成、恢复和大小。HepG2球体的形成率和恢复率均接近100%，并且随着初始细胞播种密度的增加，HepG2球体的大小也随之增加。不同微孔中球体的大小无显著差异。此外，使用TASCL装置获得的HepG2球体是活的，并产生白蛋白。利用FE-SEM研究了HepG2球体的形貌。微孔中的球体呈现完美的球形聚集。在本报告中，通过调整TASCL装置的微孔大小，可以创建均匀的HepG2球体，并且该装置可以更精确地测量每个HepG2球体的肝功能。本发明的TASCL装置可作为药物毒性评价系统。
{"title":"Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device.","authors":"Y. Miyamoto, M. Ikeuchi, H. Noguchi, T. Yagi, S. Hayashi","doi":"10.3727/215517915X689056","DOIUrl":"https://doi.org/10.3727/215517915X689056","url":null,"abstract":"In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 µm × 500 µm) and a bottom aperture (300 µm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"8 1-2 1","pages":"47-56"},"PeriodicalIF":0.0,"publicationDate":"2015-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517915X689056","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-08-01DOI: 10.3727/215517915X689083
H. Hanayama, K. Ohashi, R. Utoh, H. Shimizu, K. Ise, F. Sakurai, H. Mizuguchi, H. Tsuchiya, T. Okano, M. Gotoh
To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.
{"title":"Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.","authors":"H. Hanayama, K. Ohashi, R. Utoh, H. Shimizu, K. Ise, F. Sakurai, H. Mizuguchi, H. Tsuchiya, T. Okano, M. Gotoh","doi":"10.3727/215517915X689083","DOIUrl":"https://doi.org/10.3727/215517915X689083","url":null,"abstract":"To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"8 1-2 1","pages":"31-8"},"PeriodicalIF":0.0,"publicationDate":"2015-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517915X689083","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-04-01DOI: 10.3727/215517915X688057
Rika Nagasaki, Y. Mukudai, Y. Yoshizawa, M. Nagasaki, Sunao Shiogama, Maiko Suzuki, S. Kondo, S. Shintani, T. Shirota
The osteogenic induction of adipose-derived stem cells (ADSCs) has been regarded as an important step in bone tissue engineering. In the present study, we focused on the buccal fat pad (BFP) as a source of adipose tissue, since BFPs are encapsulated by adipose tissue and are often coextirpated during oral surgery. Low-intensity pulsed ultrasound (LIPUS) is effective in the treatment of fractures, and nanohydroxyapatite (NHA) is known as a bone substitute material. Here we investigated the synergistic effects of LIPUS and NHA in the osteogenesis of ADSCs. A combination of LIPUS irritation and NHA as a scaffold significantly increased the osteogenic differentiation of ADSCs in vitro, and in our in vivo study in which ADSCs were transplanted into calvarial bone defects of nude mice, the combinational effect greatly enhanced the new bone formation of the margin of the defects. These results demonstrate that synergistic effects of LIPUS and NHA are capable of effectively inducing the differentiation of ADSCs into osteoblasts, and they suggest a novel therapeutic strategy for bone regeneration by the autotransplantation of ADSCs.
{"title":"A Combination of Low-Intensity Pulsed Ultrasound and Nanohydroxyapatite Concordantly Enhances Osteogenesis of Adipose-Derived Stem Cells From Buccal Fat Pad.","authors":"Rika Nagasaki, Y. Mukudai, Y. Yoshizawa, M. Nagasaki, Sunao Shiogama, Maiko Suzuki, S. Kondo, S. Shintani, T. Shirota","doi":"10.3727/215517915X688057","DOIUrl":"https://doi.org/10.3727/215517915X688057","url":null,"abstract":"The osteogenic induction of adipose-derived stem cells (ADSCs) has been regarded as an important step in bone tissue engineering. In the present study, we focused on the buccal fat pad (BFP) as a source of adipose tissue, since BFPs are encapsulated by adipose tissue and are often coextirpated during oral surgery. Low-intensity pulsed ultrasound (LIPUS) is effective in the treatment of fractures, and nanohydroxyapatite (NHA) is known as a bone substitute material. Here we investigated the synergistic effects of LIPUS and NHA in the osteogenesis of ADSCs. A combination of LIPUS irritation and NHA as a scaffold significantly increased the osteogenic differentiation of ADSCs in vitro, and in our in vivo study in which ADSCs were transplanted into calvarial bone defects of nude mice, the combinational effect greatly enhanced the new bone formation of the margin of the defects. These results demonstrate that synergistic effects of LIPUS and NHA are capable of effectively inducing the differentiation of ADSCs into osteoblasts, and they suggest a novel therapeutic strategy for bone regeneration by the autotransplantation of ADSCs.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"76 1","pages":"123-31"},"PeriodicalIF":0.0,"publicationDate":"2015-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517915X688057","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-02-08DOI: 10.3727/215517914X685178
T. Tsugata, N. Nikoh, T. Kin, I. Saitoh, Yasufumi Noguchi, H. Ueki, Masami Watanabe, A. M. James Shapiro, H. Noguchi
The low efficiency of in vitro differentiation of human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs) into insulin-producing cells thus creates a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). In this study, we investigated the key factors for the differentiation of PSCs into insulin-producing cells. We obtained microarray data of HUES8 and HUES6 from two GeneChips (GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array) in a database of GEO (NCBI), since HUES8 can differentiate into pancreatic cells, while HUES6 hardly demonstrates any differentiation at all. The genes with more than fourfold higher expressions in HUES8 compared to HUES6 included RPS4Y1, DDX3Y, EIF1AY, GREM1, GATA6, and NLGN4Y. Since there were four genes, RPS4Y1, DDX3Y, EIF1AY, and NLGN4Y, on the Y chromosome and HUES8 was a male cell line and HUES6 was a female cell line, we excluded these genes in this study. On the other hand, genes with more than fourfold higher expressions in HUES6 compared to HUES8 included NLRP2, EGR1, and SMC3. We next compared iPSCs derived from pancreatic cells (PiPSCs) and iPSCs derived from fibroblasts (FiPSCs). PiPSCs differentiated into insulin-producing cells more easily than FiPSCs because of their epigenetic memory. The gene expressions of GREM1, GATA6, NLRP2, EGR1, and SMC3 in PiPSCs and FiPSCs were also investigated. The expression level of GREM1 and GATA6 in PiPSCs were higher than in FiPSCs. On the other hand, EGR1, which was lower in HUES8 than in HUES6, was predictably lower in PiPSCs than FiPSCs, while NLRP2 and SMC3 were higher in PiPSCs than FiPSCs. These data suggest that the expression of GATA6 and GREM1 and the inhibition of EGR1 may be important factors for the differentiation of PSCs into insulin-producing cells.
人类胚胎干细胞(ESCs)或人类诱导多能干细胞(iPSCs)在体外分化为胰岛素生成细胞的效率较低，因此为人类多能干细胞(PSCs)的临床应用创造了一个关键障碍。在这项研究中，我们研究了PSCs向胰岛素生成细胞分化的关键因素。我们从GEO (NCBI)数据库中的两个基因芯片(GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array)中获得了HUES8和HUES6的微阵列数据，因为HUES8可以分化为胰腺细胞，而HUES6几乎没有分化。与HUES6相比，在HUES8中表达量高出4倍以上的基因包括RPS4Y1、DDX3Y、EIF1AY、GREM1、GATA6和NLGN4Y。由于Y染色体上存在RPS4Y1、DDX3Y、EIF1AY、NLGN4Y四个基因，且HUES8为雄性细胞系，HUES6为雌性细胞系，因此我们在本研究中排除了这些基因。另一方面，与HUES8相比，在HUES6中表达量高出4倍以上的基因包括NLRP2、EGR1和SMC3。接下来，我们比较了来自胰腺细胞的iPSCs (PiPSCs)和来自成纤维细胞的iPSCs (FiPSCs)。由于具有表观遗传记忆，pipsc比fipsc更容易分化为产生胰岛素的细胞。研究了GREM1、GATA6、NLRP2、EGR1和SMC3基因在PiPSCs和FiPSCs中的表达情况。GREM1和GATA6在pipsscs中的表达水平高于fipsscs。另一方面，EGR1在HUES8中低于HUES6，在PiPSCs中低于FiPSCs，而NLRP2和SMC3在PiPSCs中高于FiPSCs。这些数据提示GATA6和GREM1的表达以及EGR1的抑制可能是PSCs向胰岛素生成细胞分化的重要因素。
{"title":"Potential Factors for the Differentiation of ESCs/iPSCs Into Insulin-Producing Cells.","authors":"T. Tsugata, N. Nikoh, T. Kin, I. Saitoh, Yasufumi Noguchi, H. Ueki, Masami Watanabe, A. M. James Shapiro, H. Noguchi","doi":"10.3727/215517914X685178","DOIUrl":"https://doi.org/10.3727/215517914X685178","url":null,"abstract":"The low efficiency of in vitro differentiation of human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs) into insulin-producing cells thus creates a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). In this study, we investigated the key factors for the differentiation of PSCs into insulin-producing cells. We obtained microarray data of HUES8 and HUES6 from two GeneChips (GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array) in a database of GEO (NCBI), since HUES8 can differentiate into pancreatic cells, while HUES6 hardly demonstrates any differentiation at all. The genes with more than fourfold higher expressions in HUES8 compared to HUES6 included RPS4Y1, DDX3Y, EIF1AY, GREM1, GATA6, and NLGN4Y. Since there were four genes, RPS4Y1, DDX3Y, EIF1AY, and NLGN4Y, on the Y chromosome and HUES8 was a male cell line and HUES6 was a female cell line, we excluded these genes in this study. On the other hand, genes with more than fourfold higher expressions in HUES6 compared to HUES8 included NLRP2, EGR1, and SMC3. We next compared iPSCs derived from pancreatic cells (PiPSCs) and iPSCs derived from fibroblasts (FiPSCs). PiPSCs differentiated into insulin-producing cells more easily than FiPSCs because of their epigenetic memory. The gene expressions of GREM1, GATA6, NLRP2, EGR1, and SMC3 in PiPSCs and FiPSCs were also investigated. The expression level of GREM1 and GATA6 in PiPSCs were higher than in FiPSCs. On the other hand, EGR1, which was lower in HUES8 than in HUES6, was predictably lower in PiPSCs than FiPSCs, while NLRP2 and SMC3 were higher in PiPSCs than FiPSCs. These data suggest that the expression of GATA6 and GREM1 and the inhibition of EGR1 may be important factors for the differentiation of PSCs into insulin-producing cells.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"7 2 1","pages":"83-93"},"PeriodicalIF":0.0,"publicationDate":"2015-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517914X685178","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-02-08DOI: 10.3727/215517914X681802
H. Takeuchi, H. Iwamoto, Yuki Nakamura, T. Hirano, O. Konno, Y. Kihara, N. Chiba, T. Yokoyama, K. Takano, T. Toraishi, K. Okuyama, C. Ikeda, Sachiko Tanaka, K. Onda, A. Soga, Yukiko Kikuchi, T. Kawaguchi, S. Kawachi, S. Unezaki, M. Shimazu
The steroid receptor (SR) complex contains FKBP51 and FKBP52, which bind to tacrolimus (TAC) and cyclophilin 40, which, in turn, bind to cyclosporine (CYA); these influence the intranuclear mobility of steroid-SR complexes. Pharmacodynamic interactions are thought to exist between steroids and calcineurin inhibitors (CNIs) on the SR complex. We examined the effect of CNIs on steroid sensitivity. Methylprednisolone (MPSL) sensitivity was estimated as the concentration inhibiting mitosis in 50% (IC50) of peripheral blood mononuclear cells and as the area under the MPSL concentration-proliferation suppressive rate curves (CPS-AUC) in 30 healthy subjects. MPSL sensitivity was compared between the additive group (AG) as the MPSL sensitivity that was a result of addition of the proliferation suppressive rate of CNIs to that of MPSL and the mixed culture group (MCG) as MPSL sensitivity of mixed culture with both MPSL and CNIs in identical patients. IC50 values of MPSL and cortisol sensitivity were examined before and 2 months after CNI administration in 23 renal transplant recipients. IC50 and CPS-AUC values of MPSL were lower in the MCG than in the AG with administration of TAC and CYA. The CPS-AUC ratio of MCG and AG was lower in the TAC group. IC50 values of MPSL and cortisol tended to be lower after administration of TAC and CYA, and a significant difference was observed in the IC50 of cortisol after TAC administration. Steroid sensitivity increased with both TAC and CYA. Furthermore, TAC had a greater effect on increasing sensitivity. Thus, concomitant administration of CNIs and steroids can increase steroid sensitivity.
{"title":"Synergistic Effects of Calcineurin Inhibitors and Steroids on Steroid Sensitivity of Peripheral Blood Mononuclear Cells.","authors":"H. Takeuchi, H. Iwamoto, Yuki Nakamura, T. Hirano, O. Konno, Y. Kihara, N. Chiba, T. Yokoyama, K. Takano, T. Toraishi, K. Okuyama, C. Ikeda, Sachiko Tanaka, K. Onda, A. Soga, Yukiko Kikuchi, T. Kawaguchi, S. Kawachi, S. Unezaki, M. Shimazu","doi":"10.3727/215517914X681802","DOIUrl":"https://doi.org/10.3727/215517914X681802","url":null,"abstract":"The steroid receptor (SR) complex contains FKBP51 and FKBP52, which bind to tacrolimus (TAC) and cyclophilin 40, which, in turn, bind to cyclosporine (CYA); these influence the intranuclear mobility of steroid-SR complexes. Pharmacodynamic interactions are thought to exist between steroids and calcineurin inhibitors (CNIs) on the SR complex. We examined the effect of CNIs on steroid sensitivity. Methylprednisolone (MPSL) sensitivity was estimated as the concentration inhibiting mitosis in 50% (IC50) of peripheral blood mononuclear cells and as the area under the MPSL concentration-proliferation suppressive rate curves (CPS-AUC) in 30 healthy subjects. MPSL sensitivity was compared between the additive group (AG) as the MPSL sensitivity that was a result of addition of the proliferation suppressive rate of CNIs to that of MPSL and the mixed culture group (MCG) as MPSL sensitivity of mixed culture with both MPSL and CNIs in identical patients. IC50 values of MPSL and cortisol sensitivity were examined before and 2 months after CNI administration in 23 renal transplant recipients. IC50 and CPS-AUC values of MPSL were lower in the MCG than in the AG with administration of TAC and CYA. The CPS-AUC ratio of MCG and AG was lower in the TAC group. IC50 values of MPSL and cortisol tended to be lower after administration of TAC and CYA, and a significant difference was observed in the IC50 of cortisol after TAC administration. Steroid sensitivity increased with both TAC and CYA. Furthermore, TAC had a greater effect on increasing sensitivity. Thus, concomitant administration of CNIs and steroids can increase steroid sensitivity.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"7 2 1","pages":"51-7"},"PeriodicalIF":0.0,"publicationDate":"2015-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517914X681802","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-02-08DOI: 10.3727/215517914X685196
H. Noguchi
On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Professor Paul R. Sanberg (Department of Neurosurgery, College of Medicine, University of South Florida, FL, USA), Executive Editor of Cell Medicine, for providing us such an excellent opportunity to publish the data that were presented at the annual meeting of JSOPMB. I also thank Dr. David Eve, Associate Editor of Cell Medicine, for editing of our papers in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to newly join this organization. To answer the current problem of the severe human donor organ shortage for cell therapies is a big challenge. Research on adult and embryonic stem cells and artificial cell development, in addition to the recent and rapidly evolving invention of induced pluripotent stem cells, encourages us to address the problems confronting cell transplantation. Therefore, JSOPMB has now importantly focused on regenerative medicine in collaboration with cell biologists. One of the extremely important missions of the annual meeting of JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 700 members and is run under the direction of Dr. Takehide Asano, the President of JSOPMB. Excellent presentations conducted at the 40th annual meeting of JSOPMB held November 9–10, 2013, in Tokyo, Japan, under the supervision of Dr. Motohide Shimazu (Professor, Department of Gastroenterological Surgery and Transplant Surgery, Tokyo Medical University, Tokyo, Japan) were selected and given an opportunity to be published in this special issue of Cell Medicine. Five of these presentations are herein published in this special JSOPMB issue. Takeuchi et al. reported on the synergistic effects of calcineurin inhibitors [tacrolimus (TAC), cyclosporine (CYA)] and steroids on steroid sensitivity of peripheral blood mononuclear cells in humans. Steroid sensitivity was observed to increase with both TAC and CYA. Sufiandi et al. showed the effect of shear stress on rat hepatocyte viability under horizontal and vertical syringe orientation. The vertical syringe orientation resulted in lower viability loss than the horizontal orientation, suggesting that removing a sedimentation effect is important to improving cell viability by preventing high shear stress. Miyamoto et al. showed three-dimensional in vitro hepatic constructs formed using a combinatorial tapered stencil for cluster cultu
我谨代表日本器官保存与医学生物学学会(JSOPMB)向《细胞医学》杂志的执行编辑Paul R. Sanberg教授(美国佛罗里达州南佛罗里达大学医学院神经外科)表示衷心的感谢，感谢他为我们提供了这样一个绝佳的机会来发表在JSOPMB年会上提交的数据。我还要感谢《细胞医学》副主编David Eve博士对我们论文的详细编辑。我非常确信，细胞医学与JSOPMB之间的关系增强了JSOPMB成员和董事会成员的动力，并将在未来继续这样做，同时也鼓励年轻的日本研究人员加入这个组织。解决目前细胞治疗中严重的人类供体器官短缺问题是一个巨大的挑战。成体和胚胎干细胞和人工细胞发育的研究，以及最近和快速发展的诱导多能干细胞的发明，鼓励我们解决细胞移植面临的问题。因此，JSOPMB现在与细胞生物学家合作，重点关注再生医学。JSOPMB年会的一个极其重要的任务是交流新的研究成果和创造新的治疗理念。JSOPMB一直鼓励和激励年轻的调查人员。JSOPMB成立于1974年，目的是研究器官保存，在20世纪90年代得到了医学、药理学、工程、兽医学和基础科学等各个领域的研究人员的广泛发展。目前，JSOPMB有700多名成员，并在JSOPMB主席浅野武雄博士的指导下运作。在Motohide Shimazu博士(东京医科大学胃肠外科和移植外科教授，日本东京)的监督下，于2013年11月9日至10日在日本东京举行的第40届JSOPMB年会上进行的优秀报告被选中并有机会发表在这期《细胞医学》特刊上。其中的五个演讲将在本期JSOPMB特刊中发表。Takeuchi等人报道了钙调磷酸酶抑制剂[他克莫司(TAC)、环孢素(CYA)]和类固醇对人外周血单个核细胞类固醇敏感性的协同作用。类固醇敏感性随TAC和CYA的增加而增加。Sufiandi等研究了水平和垂直注射方向下剪切应力对大鼠肝细胞活力的影响。与水平方向相比，垂直注射器方向导致的活力损失更低，这表明消除沉淀效应对于通过防止高剪切应力来提高细胞活力很重要。Miyamoto等人展示了使用组合锥形支架进行集群培养(TASCL)装置形成的三维体外肝脏结构。采用塑料培养皿作为组合TASCL装置的底部，通过增加原代小鼠肝细胞的播种细胞密度，获得大小均匀的三维肝细胞构建体。Yukawa等人利用量子点研究了活体自身荧光对体内荧光成像的影响。使用Maestro™体内成像系统，使用各种激发/荧光滤光片(蓝色、绿色、黄色、红色、深红色和近红外)检测脱毛小鼠的背侧和腹侧自身荧光。然而，在红色滤光条件下，可以检测到移植的脂肪组织源性干细胞在小鼠背部标记的量子点，这表明使用量子点的荧光成像可以用于移植细胞的标记和检测。Tsugata等人利用微阵列数据和来自人胰岛或成纤维细胞的诱导多能干细胞(iPSCs)研究了多能干细胞向胰岛素生成细胞分化的关键因素。他们的数据表明，鸟嘌呤-腺嘌呤-胸腺嘧啶-腺嘌呤结合蛋白6 (GATA6)和格林林1 (GREM1)的表达以及早期生长反应1 (EGR1)的抑制可能是PSCs向胰岛素生成细胞分化的重要因素。这期JSOPMB的主题是“器官生物学的基础和临床科学”。董事会成员和我期待着JSOPMB与细胞医学联合取得进一步进展。
{"title":"Basic and Clinical Science for Organ Biology.","authors":"H. Noguchi","doi":"10.3727/215517914X685196","DOIUrl":"https://doi.org/10.3727/215517914X685196","url":null,"abstract":"On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Professor Paul R. Sanberg (Department of Neurosurgery, College of Medicine, University of South Florida, FL, USA), Executive Editor of Cell Medicine, for providing us such an excellent opportunity to publish the data that were presented at the annual meeting of JSOPMB. I also thank Dr. David Eve, Associate Editor of Cell Medicine, for editing of our papers in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to newly join this organization. \u0000 \u0000To answer the current problem of the severe human donor organ shortage for cell therapies is a big challenge. Research on adult and embryonic stem cells and artificial cell development, in addition to the recent and rapidly evolving invention of induced pluripotent stem cells, encourages us to address the problems confronting cell transplantation. Therefore, JSOPMB has now importantly focused on regenerative medicine in collaboration with cell biologists. \u0000 \u0000One of the extremely important missions of the annual meeting of JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 700 members and is run under the direction of Dr. Takehide Asano, the President of JSOPMB. \u0000 \u0000Excellent presentations conducted at the 40th annual meeting of JSOPMB held November 9–10, 2013, in Tokyo, Japan, under the supervision of Dr. Motohide Shimazu (Professor, Department of Gastroenterological Surgery and Transplant Surgery, Tokyo Medical University, Tokyo, Japan) were selected and given an opportunity to be published in this special issue of Cell Medicine. Five of these presentations are herein published in this special JSOPMB issue. \u0000 \u0000Takeuchi et al. reported on the synergistic effects of calcineurin inhibitors [tacrolimus (TAC), cyclosporine (CYA)] and steroids on steroid sensitivity of peripheral blood mononuclear cells in humans. Steroid sensitivity was observed to increase with both TAC and CYA. \u0000 \u0000Sufiandi et al. showed the effect of shear stress on rat hepatocyte viability under horizontal and vertical syringe orientation. The vertical syringe orientation resulted in lower viability loss than the horizontal orientation, suggesting that removing a sedimentation effect is important to improving cell viability by preventing high shear stress. \u0000 \u0000Miyamoto et al. showed three-dimensional in vitro hepatic constructs formed using a combinatorial tapered stencil for cluster cultu","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"7 2 1","pages":"49"},"PeriodicalIF":0.0,"publicationDate":"2015-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517914X685196","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756263","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-01-01DOI: 10.3727/215517914X685187
Y. Miyamoto, M. Ikeuchi, H. Noguchi, T. Yagi, S. Hayashi
Attempts to create artificial liver tissue from various cells have been reported as an alternative method for liver transplantation and pharmaceutical testing. In the construction of artificial liver tissue, the selection of the cell source is the most important factor. However, if an appropriate environment (in vitro/in vivo) cannot be provided for various cells, it is not possible to obtain artificial liver tissue with the desired function. Therefore, we focused on the in vitro environment and produced liver tissues using MEMS technology. In the present study, we report a combinatorial TASCL device to prepare 3D cell constructs in vitro. The TASCL device was fabricated with an overall size of 10 mm × 10 mm with microwells and a top aperture (400 µm × 400 µm, 600 µm × 600 µm, 800 µm × 800 µm) and bottom aperture (40 µm × 40 µm, 80 µm × 80 µm, 160 µm × 160 µm) per microwell. The TASCL device can be easily installed on various culture dishes with tweezers. Using plastic dishes as the bottom surface of the combinatorial TASCL device, 3D hepatocyte constructs of uniform sizes (about ɸ 100 μm-ɸ 200 μm) were produced by increasing the seeding cell density of primary mouse hepatocytes. The 3D hepatocyte constructs obtained using the TASCL device were alive and secreted albumin. On the other hand, partially adhered primary mouse hepatocytes exhibited a cobblestone morphology on the collagen-coated bottom of the individual microwells using the combinatorial TASCL device. By changing the bottom substrate of the TASCL device, the culture environment of the cell constructs was easily changed to a 3D environment. The combinatorial TASCL device described in this report can be used quickly and simply. This device will be useful for preparing hepatocyte constructs for application in drug screening and cell medicine.
{"title":"Three-Dimensional In Vitro Hepatic Constructs Formed Using Combinatorial Tapered Stencil for Cluster Culture (TASCL) Device.","authors":"Y. Miyamoto, M. Ikeuchi, H. Noguchi, T. Yagi, S. Hayashi","doi":"10.3727/215517914X685187","DOIUrl":"https://doi.org/10.3727/215517914X685187","url":null,"abstract":"Attempts to create artificial liver tissue from various cells have been reported as an alternative method for liver transplantation and pharmaceutical testing. In the construction of artificial liver tissue, the selection of the cell source is the most important factor. However, if an appropriate environment (in vitro/in vivo) cannot be provided for various cells, it is not possible to obtain artificial liver tissue with the desired function. Therefore, we focused on the in vitro environment and produced liver tissues using MEMS technology. In the present study, we report a combinatorial TASCL device to prepare 3D cell constructs in vitro. The TASCL device was fabricated with an overall size of 10 mm × 10 mm with microwells and a top aperture (400 µm × 400 µm, 600 µm × 600 µm, 800 µm × 800 µm) and bottom aperture (40 µm × 40 µm, 80 µm × 80 µm, 160 µm × 160 µm) per microwell. The TASCL device can be easily installed on various culture dishes with tweezers. Using plastic dishes as the bottom surface of the combinatorial TASCL device, 3D hepatocyte constructs of uniform sizes (about ɸ 100 μm-ɸ 200 μm) were produced by increasing the seeding cell density of primary mouse hepatocytes. The 3D hepatocyte constructs obtained using the TASCL device were alive and secreted albumin. On the other hand, partially adhered primary mouse hepatocytes exhibited a cobblestone morphology on the collagen-coated bottom of the individual microwells using the combinatorial TASCL device. By changing the bottom substrate of the TASCL device, the culture environment of the cell constructs was easily changed to a 3D environment. The combinatorial TASCL device described in this report can be used quickly and simply. This device will be useful for preparing hepatocyte constructs for application in drug screening and cell medicine.","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"7 2 1","pages":"67-74"},"PeriodicalIF":0.0,"publicationDate":"2015-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517914X685187","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2014-12-12eCollection Date: 2015-02-08DOI: 10.3727/215517914X685169
Hiroshi Yukawa, Masaki Watanabe, Noritada Kaji, Yoshinobu Baba
Quantum dots (QDs) are thought to be a novel inorganic probe for in vivo fluorescence imaging because of their excellent fluorescence properties. Autofluorescence is generally known to be produced from various living bodies including humans, rats, and mice. However, the influence of the autofluorescence on in vivo fluorescence imaging using QDs remains poorly understood. In this article, we assessed the autofluorescence derived from a mouse body and the influence of the autofluorescence on in vivo fluorescence imaging using QDs. The dorsal and ventral autofluorescence derived from a mouse from which the hair was removed were detected under all kinds of excitation/fluorescence filter settings (blue, green, yellow, red, deep red, and NIR) using the Maestro™ in vivo imaging system. The degree of autofluorescence was found to be extremely high in the red filter condition, but transplanted ASCs labeled with QDs on the back of a mouse could be detected in the red filter condition. Moreover, the ASCs labeled with QDs could be traced for at least 5 days. We suggest that fluorescence imaging using QDs can be useful for the detection of transplanted cells.
{"title":"Influence of Autofluorescence Derived From Living Body on In Vivo Fluorescence Imaging Using Quantum Dots.","authors":"Hiroshi Yukawa, Masaki Watanabe, Noritada Kaji, Yoshinobu Baba","doi":"10.3727/215517914X685169","DOIUrl":"10.3727/215517914X685169","url":null,"abstract":"<p><p>Quantum dots (QDs) are thought to be a novel inorganic probe for in vivo fluorescence imaging because of their excellent fluorescence properties. Autofluorescence is generally known to be produced from various living bodies including humans, rats, and mice. However, the influence of the autofluorescence on in vivo fluorescence imaging using QDs remains poorly understood. In this article, we assessed the autofluorescence derived from a mouse body and the influence of the autofluorescence on in vivo fluorescence imaging using QDs. The dorsal and ventral autofluorescence derived from a mouse from which the hair was removed were detected under all kinds of excitation/fluorescence filter settings (blue, green, yellow, red, deep red, and NIR) using the Maestro™ in vivo imaging system. The degree of autofluorescence was found to be extremely high in the red filter condition, but transplanted ASCs labeled with QDs on the back of a mouse could be detected in the red filter condition. Moreover, the ASCs labeled with QDs could be traced for at least 5 days. We suggest that fluorescence imaging using QDs can be useful for the detection of transplanted cells. </p>","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"7 2 1","pages":"75-82"},"PeriodicalIF":0.0,"publicationDate":"2014-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Improving cell viability and function are important for enhancing the clinical results of cell transplantation. The relationship between cell viability and shear stress remains unexplained, and sedimentation effects during the infusion process are important to the hepatocyte transplantation process. In the present study, the relationship between cell viability and shear stress in the presence of sedimentation effect was investigated using a microchannel simulating the cell transplantation process under several shear stress conditions. Horizontal and vertical syringe orientations were employed to investigate the sedimentation effect. The vertical syringe orientation resulted in lower viability loss than the horizontal orientation. In summary, removing a sedimentation effect is important to improving cell viability by preventing high shear stress.
{"title":"Improvement of Infusion Process in Cell Transplantation: Effect of Shear Stress on Hepatocyte Viability Under Horizontal and Vertical Syringe Orientation.","authors":"Sandi Sufiandi, Hiromichi Obara, Shin Enosawa, Huai-Che Hsu, Naoto Matsuno, Hiroshi Mizunuma","doi":"10.3727/215517914X685150","DOIUrl":"10.3727/215517914X685150","url":null,"abstract":"<p><p>Improving cell viability and function are important for enhancing the clinical results of cell transplantation. The relationship between cell viability and shear stress remains unexplained, and sedimentation effects during the infusion process are important to the hepatocyte transplantation process. In the present study, the relationship between cell viability and shear stress in the presence of sedimentation effect was investigated using a microchannel simulating the cell transplantation process under several shear stress conditions. Horizontal and vertical syringe orientations were employed to investigate the sedimentation effect. The vertical syringe orientation resulted in lower viability loss than the horizontal orientation. In summary, removing a sedimentation effect is important to improving cell viability by preventing high shear stress. </p>","PeriodicalId":9780,"journal":{"name":"Cell medicine","volume":"7 2 1","pages":"59-66"},"PeriodicalIF":0.0,"publicationDate":"2014-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3727/215517914X685150","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69756627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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