Co-reporter:Toshiki Matsui, Yusuke Arima, Naohiro Takemoto, Hiroo Iwata
Acta Biomaterialia 2015 Volume 13() pp:32-41
Publication Date(Web):February 2015
DOI:10.1016/j.actbio.2014.11.011
Abstract
Polylactic acid (PLA) is a candidate material to prepare scaffolds for 3-D tissue regeneration. However, cells do not adhere or proliferate well on the surface of PLA because it is hydrophobic. We report a simple and rapid method for inducing cell adhesion to PLA through DNA hybridization. Single-stranded DNA (ssDNA) conjugated to poly(ethylene glycol) (PEG) and to a terminal phospholipid (ssDNA-PEG-lipid) was used for cell surface modification. Through DNA hybridization, modified cells were able to attach to PLA surfaces modified with complementary sequence (ssDNA′). Different cell types can be attached to PLA fibers and films in a spatially controlled manner by using ssDNAs with different sequences. In addition, they proliferate well in a culture medium supplemented with fetal bovine serum. The coexisting modes of cell adhesion through DNA hybridization and natural cytoskeletal adhesion machinery revealed no serious effects on cell growth. The combination of a 3-D scaffold made of PLA and cell immobilization on the PLA scaffold through DNA hybridization will be useful for the preparation of 3-D tissue and organs.
Co-reporter:Toru Itagaki, Yusuke Arima, Rei Kuwabara, Narufumi Kitamura, Hiroo Iwata
Colloids and Surfaces B: Biointerfaces 2015 Volume 135() pp:765-773
Publication Date(Web):1 November 2015
DOI:10.1016/j.colsurfb.2015.08.014
•Interaction of PEG-lipids carrying different lipid parts with cells were examined.•Cell membrane was modified with PEG-lipids within 15 min without cytotoxicity.•PEG-lipid densities on cell surface ranged from 10−3 to 10−2 molecules/nm2.•Hydrophobicity of lipid part affects dissociation rate but not anchoring rate.Eight types of poly(ethylene glycol)-lipid(PEG-lipids) carrying different lipid tails were synthesized. These PEG-lipids were labeled with fluorescein isothiocyanate (FITC-PEG-lipids) to examine their interaction with cells and to quantitatively determine amounts of PEG-lipids bound on the cell surface. FITC-PEG-lipids spontaneously anchored to the cell membrane within 15 min without loss of cell viability. The type of lipid had very little effect on the anchoring rates, while an increase in the hydrophobicity of the lipid portion of the PEG-lipids slowed their dissociation rates. Densities of FITC-PEG-lipids on the cell surface ranged from 1 × 10−3 to 1 × 10−2 molecules/nm2, depending on the kinds of lipids employed. The relationship between the stability of the lipids on the cell membrane and the hydrophobicity of the lipid moieties will give a basis for the selection of a hydrophobic moiety in PEG-lipid conjugates for use in specific applications.
Co-reporter:Ian T. Hoffecker, Naohiro Takemoto, Yusuke Arima, Hiroo Iwata
Biomaterials 2015 53() pp: 318-329
Publication Date(Web):
DOI:10.1016/j.biomaterials.2015.02.059
Co-reporter:Sho Deno, Naohiro Takemoto, Hiroo Iwata
Bioorganic & Medicinal Chemistry 2014 Volume 22(Issue 1) pp:350-357
Publication Date(Web):1 January 2014
DOI:10.1016/j.bmc.2013.11.023
Ischemia–reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)–phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of complementary deoxythymidylic acid (oligo(dT)20), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)20 and oligo(dT)20. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.Immobilization of fluorescent liposomes on HUVEC surfaces via DNA hybridization and its introduction into cytoplasm.
Co-reporter:Mitsuaki Toda, Yusuke Arima, Hiromi Takiguchi, Hiroo Iwata
Analytical Biochemistry 2014 Volume 467() pp:47-53
Publication Date(Web):15 December 2014
DOI:10.1016/j.ab.2014.08.029
Co-reporter:Tadashi Nakaji-Hirabayashi, Koichi Kato, and Hiroo Iwata
Bioconjugate Chemistry 2013 Volume 24(Issue 11) pp:1798
Publication Date(Web):August 31, 2013
DOI:10.1021/bc400005m
Poor viability of cells transplanted into the brain has been the critical problem associated with stem cell-based therapy for Parkinson’s disease. To overcome this problem, a collagen hydrogel incorporating an integrin-binding protein complex was prepared and used as a carrier for neural stem cells. The protein complex consisted of two polypeptides containing the G3 domain of a laminin α1 chain and the C-terminal oligopeptide of a laminin γ1 chain. These polypeptides were fused with α-helical segments which spontaneously formed a coiled-coil heterodimer and with the collagen-binding peptide that facilitated the binding of the heterodimer to collagen networks. In this study, neural stem cells stably expressing the enhanced green fluorescent protein (EGFP) were suspended in the hydrogel and transplanted into the striatum of healthy rats. The viability of transplanted cells was evaluated by histological analysis and quantitative reverse-transcriptase polymerase chain reaction for EGFP mRNA present in the tissue explants. Our results showed that the collagen hydrogel incorporating the integrin-binding protein complex serves to improve the viability of neural stem cells (NSCs) in the early stage after transplantation into the striatum.
Co-reporter:Naohiro Takemoto, Yuji Teramura and Hiroo Iwata
Biomaterials Science 2013 vol. 1(Issue 3) pp:315-321
Publication Date(Web):28 Nov 2012
DOI:10.1039/C2BM00048B
Sertoli cells play a crucial role in creating the immunoprivileged environment of the testis. We examined the survival of islets of Langerhans after co-transplantation with Sertoli cells. Sertoli cells near islets should protect the graft from rejection. In this study, conjugates of single stranded oligonucleotides, poly(ethylene glycol) and phospholipids (ssDNA-PEG-DPPE) were used to immobilize Sertoli cells on islets. The 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of deoxythymidylic acid (oligo(dT)20) were presented as ssDNAs on the surfaces of Sertoli cells and islets, respectively, through the hydrophobic interaction between a lipid unit of the conjugates and the cell membrane. The Sertoli cells were immobilized on the islets through hybridization between oligo(dA)20 and oligo(dT)20. When Sertoli cell-immobilized islets were infused into the liver of mice through the portal vein, the Sertoli cells remained around the islets.
Co-reporter:Narufumi Kitamura, Ryusuke Nakai, Haruyasu Kohda, Keiko Furuta-Okamoto, Hiroo Iwata
Bioorganic & Medicinal Chemistry 2013 Volume 21(Issue 22) pp:7175-7181
Publication Date(Web):15 November 2013
DOI:10.1016/j.bmc.2013.08.063
A labeling method for islet cells with superparamagnetic iron oxide nanoparticles (SPIOs) based on DNA hybridization is proposed for monitoring of transplanted islets by magnetic resonance imaging (MRI). The surfaces of SPIOs were modified by via Michael reaction by reacting oligo-(deoxyadenylic acid)-bearing a terminal thiol group at the 5′-end ((dA)20-SH) with maleic acid functional groups on the SPIOs. The SPIOs were immobilized on islet cells which had been pretreated with oligo-(thymidylic acid)–poly(ethylene glycol)–phospholipid conjugates ((dT)20–PEG–DPPE) through DNA hybridization. Transmission electron microscopy observations revealed that SPIOs were initially anchored on the islet cell surfaces and subsequently transferred to endosomes or exfoliated with time. The SPIO-labeled islet cells could be clearly detected as dark spots by T2∗-weighted MR image, whereas non-labeled islet cells could not be detected.DNA hybridization is proposed for monitoring of transplanted islets by magnetic resonance imaging (MRI). The surfaces of SPIOs were modified with oligo-(deoxyadenylic acid) ((dA)20). The SPIOs were immobilized on islet cells which had been pretreated with oligo-(thymidylic acid)–poly(ethylene glycol)–phospholipid conjugates ((dT)20–PEG–DPPE) through DNA hybridization. Transmission electron microscopy observations revealed that SPIOs were initially anchored on the islet cell surfaces and subsequently transferred to endosomes with time. The SPIO-labeled islet cells could be clearly detected as dark spots by T2-weighted MR image.
Co-reporter:Nguyen Minh Luan, Hiroo Iwata
Biomaterials 2013 34(21) pp: 5019-5024
Publication Date(Web):
DOI:10.1016/j.biomaterials.2013.03.041
Co-reporter:Kengo Sakurai, Ian T. Hoffecker, Hiroo Iwata
Biomaterials 2013 34(2) pp: 361-370
Publication Date(Web):
DOI:10.1016/j.biomaterials.2012.09.080
Co-reporter:Tomonobu Kodama
Journal of Biomedical Materials Research Part B: Applied Biomaterials 2013 Volume 101B( Issue 4) pp:656-662
Publication Date(Web):
DOI:10.1002/jbm.b.32869
Abstract
Endovascular treatment of intracranial aneurysms with detachable coils has been accepted widely. Problems of coil compaction, recanalization and rare endothelialization at the aneurysm orifice are not yet solved. We investigated the efficacy of a simvastatin coating applied without any additional matrix to coils to accelerate thrombus organization in the cavity in a rat model of aneurysm. Twelve metal coils coated with simvastatin and 12 bare coils were inserted into the ligated external carotid arterial (ECA) sacs of rats. The ECA sacs were removed 2 or 4 weeks after the coils were implanted and examined by histology and immunohistochemical assay. The organized areas in the ECA sacs in the simvastatin group (73.6 ± 19.4%, 2 wk; 83.4 ± 11.1%, 4 wk) was significantly higher p = 0.003, 2 wk; p = 0.0004, 4 wk than the bare metal group at 2 and 4 weeks (20.5 ± 10.7%, 2 wk, p < 0.003; 37.4 ± 20.6%, 4 wk, p < 0.0004). Organized tissues that formed around the coils coated with simvastatin were characterized by an accumulation of cells positive for αSMA and collagen connective matrix. Tissues also were accompanied by marked formation of endothelium at the orifice of the ECA sac. We suggest that coating coils with simvastatin effectively accelerated organization within the aneurysms and endothelialization over the coil. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part 2013.
Co-reporter:Tadashi Nakaji-Hirabayashi, Koichi Kato, and Hiroo Iwata
Bioconjugate Chemistry 2012 Volume 23(Issue 2) pp:212
Publication Date(Web):January 10, 2012
DOI:10.1021/bc200481v
Cell transplantation is a potential methodology for the treatment of Parkinson’s disease. However, the therapeutic effect is limited by poor viability of transplanted cells. To overcome this problem, we hypothesized that a dual step approach, whereby providing an adhesive substrate for transplanted cells and, at the same time, by preventing the infiltration of activated microglia into the site of transplantation promotes the cell survival. To establish above conditions, attempts were made to prepare 3-D matrices using collagen hydrogels that incorporated integrin-binding polypeptides derived from laminin-1. Tandem combinations of laminin globular domains as well as a single globular domain 3 were prepared using recombinant DNA technology as a fusion with hexahistidine and bound to metal chelated surfaces to screen for the adhesion and proliferation of neural stem cells (NSCs). In addition, a small peptide derived from laminin γ1 chain was prepared and heterodimerized with the globular domain-containing chimeric proteins to evaluate for the enhancement of integrin-mediated cell adhesion. As a result, a heterodimer consisting of the globular domain 3 of the laminin α1 chain and the peptide from the laminin γ1 chain was selected as the best candidate among the polypeptides studied here for the incorporation into a collagen hydrogel. It was shown that the survival of NSCs was indeed promoted in the collagen hydrogel incorporating the heterodimer compared to the pure collagen hydrogel.
Co-reporter:Takashi Murakami, Yusuke Arima, Mitsuaki Toda, Hiromi Takiguchi, Hiroo Iwata
Analytical Biochemistry 2012 Volume 421(Issue 2) pp:632-639
Publication Date(Web):15 February 2012
DOI:10.1016/j.ab.2011.12.008
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) combines enhanced field platform and fluorescence detection. Its advantages are the strong intensity of the electromagnetic field and the high signal/noise (S/N) ratio due to the localized evanescent field at the water/metal interface. However, the energy transfer from the fluorophore to the metal surface diminishes the fluorescence intensity, and this reduces the sensitivity. In this study, we tested whether polystyrene (PSt) could act as a dielectric layer to suppress the energy transfer from the fluorophore to the metal surface. We hypothesized that this would improve the sensitivity of SPFS-based immunoassays. We used α-fetoprotein (AFP) as a model tumor biomarker in the sandwich-type immunoassay. We determined the relationship between fluorescent signal intensity and PSt layer thickness and compared this to theoretical predictions. We found that the fluorescence signal increased by optimally controlling the thickness of the PSt layer. Our results indicated that the SPFS-based immunoassay is a promising clinical diagnostic tool for quantitatively determining the concentrations of low-level biomarkers in blood samples.
Co-reporter:Nguyen Minh Luan, Hiroo Iwata
Biomaterials 2012 33(32) pp: 8075-8081
Publication Date(Web):
DOI:10.1016/j.biomaterials.2012.07.048
Co-reporter:Hao Chen, Yuji Teramura, Hiroo Iwata
Journal of Controlled Release 2011 Volume 150(Issue 2) pp:229-234
Publication Date(Web):10 March 2011
DOI:10.1016/j.jconrel.2010.11.011
Transplantation of islets of Langerhans is a promising method for treating patients with insulin-dependent diabetes mellitus. The major obstacle in clinical settings is early graft loss due to inflammation triggered by blood coagulation and complement activation on the surface of the islets after intraportal transplantation. We propose a versatile method for modifying the surface of islets with the fibrinolytic enzyme urokinase and the soluble domain of the anticoagulant enzyme thrombomodulin. The surfaces of islets were modified with a poly(ethylene glycol)–phospholipid conjugate bearing a maleimide group (Mal–PEG–lipid; PEG MW = 5000 kDa). The Mal–PEG–lipid anchored to the cell membranes of islets, resulting in the presentation of functional maleimide groups on the islet surface. The surface was further treated with thiolated urokinase and thrombomodulin that conjugated by thiol/maleimide bonding. No practical islet volume increase was observed after surface modification, and the modifications did not impair insulin release in response to glucose stimulation. Furthermore, the activity of the immobilized urokinase and thrombomodulin was maintained. These modifications could help to improve graft survival by preventing thrombus formation on the surface of transplanted islets.
Co-reporter:Naohiro Takemoto, Yuji Teramura, and Hiroo Iwata
Bioconjugate Chemistry 2011 Volume 22(Issue 4) pp:673
Publication Date(Web):March 9, 2011
DOI:10.1021/bc100453r
Transplantation of islets of Langerhans (islets) has been proposed as a safe, effective approach to treating patients with insulin-dependent diabetes mellitus (type I diabetes). It has been reported, however, that many islets are lost in the early phase after intraportal transplantation by instant blood coagulation-mediated inflammatory reactions. In this study, DNA hybridization was applied to conjugate the fibrinolytic enzyme urokinase on the islet surface. We synthesized amphiphilic polymers, PEG-lipids carrying oligo(dT)20 (oligo(dT)20−PEG-lipid; PEG MW = 5000) and urokinase (UK) carrying oligo(dA)20. The oligo(dT)20−PEG-lipid was spontaneously incorporated into the cell membrane through interactions between the hydrophobic parts of the PEG-lipids and the lipid bilayer, and UK was conjugated on the cell surface through DNA hybridization between oligo(dT)20 on the cell and complementary oligo(dA)20 on the UK. The activity of UK was maintained on the islet surface. The surface modification with UK did not influence islet morphology or islet ability to secrete insulin in response to changes in glucose concentration. No practical volume increase was observed with our method, indicating that islet graft loss could be suppressed at the early stage of intraportal islet transplantation.
Co-reporter:Hao Chen, Yuji Teramura, Hiroo Iwata
Biomaterials 2011 32(31) pp: 7971-7977
Publication Date(Web):
DOI:10.1016/j.biomaterials.2011.07.002
Co-reporter:Nobuharu Hisano;Yuji Teramura;Hao Chen;Yoshito Ikada
Journal of Polymer Science Part A: Polymer Chemistry 2011 Volume 49( Issue 3) pp:671-679
Publication Date(Web):
DOI:10.1002/pola.24478
Abstract
Kinetic analyses were carried out for formation of disulfide crosslinkages between thiol groups on linear polymers, poly(acrylamide-co-N-acrylcysteamine) (P-SH). Disulfide crosslinkages were formed by auto-oxidation of pendant thiol groups or through the thiol-disulfide exchange reaction induced by addition of disulfide compounds gluthathione. In the auto-oxidation reaction, the rate constant for disulfide formation highly depended on pH values of the reaction mixtures and the P-SH concentrations. Gelation rate is too slow to enclose living cells into hydrogel under physiological pH 7.4. The hydrogel formation rate can be accelerated by addition of disulfides, such as oxidized glutathione. In the later case, oxygen in the reaction mixture is not consumed. The thiol-disulfide exchange reaction is much more suitable for the cell encapsulation than the thiol auto-oxidation reaction. These findings give a basis for enclosure of living cells in a hydrogel. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011
Co-reporter:Nguyen Minh Luan, Yuji Teramura, Hiroo Iwata
Biomaterials 2011 Volume 32(Issue 27) pp:6487-6492
Publication Date(Web):September 2011
DOI:10.1016/j.biomaterials.2011.05.048
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.
Co-reporter:Kengo Sakurai, Yuji Teramura, Hiroo Iwata
Biomaterials 2011 32(14) pp: 3596-3602
Publication Date(Web):
DOI:10.1016/j.biomaterials.2011.01.066
Co-reporter:Nguyen M. Luan, Yuji Teramura, Hiroo Iwata
Biomaterials 2011 32(20) pp: 4539-4545
Publication Date(Web):
DOI:10.1016/j.biomaterials.2011.03.017
Co-reporter:Mitsuaki Toda and Hiroo Iwata
ACS Applied Materials & Interfaces 2010 Volume 2(Issue 4) pp:1107
Publication Date(Web):April 9, 2010
DOI:10.1021/am900891h
Some studies have demonstrated that amino groups, acting as nucleophiles, are potent activators of the complement system, but others not. To clarify these contradictory results, we examined complement activation on two series of NH2/CH3 and NH2/COOH mixed self-assembled monolayers (SAMs). NH2/CH3 mixed SAMs were not potent activators of the complement system regardless of the ratio of NH2/CH3 in mixed SAMs. Numerous serum proteins, such as albumin, were adsorbed onto those SAMs and formed a protein layer which inhibited access of C3b to amino groups. In contrast, much C3b and/or C3bBb were deposited on NH2/COOH mixed SAMs with ∼50−60% NH2 density on the surface and SC5b-9 was found in serum exposed to this SAM, indicating activation of the complement system. These results suggest that C3b can easily access nucleophilic NH2 groups because of the decrease in electrostatic interaction between negatively charged proteins and the NH2 SAM surface.Keywords: blood compatibility; complement activation; protein adsorption; self-assembled monolayers
Co-reporter:Osamu Inui, Yuji Teramura and Hiroo Iwata
ACS Applied Materials & Interfaces 2010 Volume 2(Issue 5) pp:1514
Publication Date(Web):May 7, 2010
DOI:10.1021/am100134v
We tested two kinds of amphiphilic polymers for cell surface modification: a poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) and a poly(vinyl alcohol) that carried alkyl side chains (PVA-alkyl). Both polymers were expected to anchor to the lipid bilayer of the cell membrane through hydrophobic interactions. We followed the kinetics of these fluorescently labeled amphiphilic polymers (fPEG-lipid, fPVA-alkyl) over time on living cells with confocal scanning laser microscopy and flow cytometry. We found that fPEG-lipids and fPVA-alkyl polymers were not cytotoxic, and they were released from the cell surface without triggering endocytosis. The gradual release from the cell surface was influenced by the hydrophobicity of the alkyl chains, which affected their stability. The amphiphilic polymers tended to aggregate on the cell surface; in particular, the aggregation of PVA-alkyl was clearly identified. Although most of PEG-lipids and PVA-alkyl polymers did not appear to in the cytoplasm, the cells were able to endocytose lipid molecules, as expected. These results suggested that the retention time of modified amphiphilic polymers on the cell surface should be a consideration when modifying cell surfaces to enhance cell transplantation.Keywords: amphiphilic polymers; cell transplantation; poly(ethylene glycol)-conjugated phospholipid (PEG-lipid); poly(vinyl alcohol); surface modification
Co-reporter:Mitsuaki Toda, Yusuke Arima, Hiroo Iwata
Acta Biomaterialia 2010 Volume 6(Issue 7) pp:2642-2649
Publication Date(Web):July 2010
DOI:10.1016/j.actbio.2010.01.035
Abstract
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 α-mercaptoethyl-ω-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.
Co-reporter:Yuji Teramura, Luan Nguyen Minh, Takuo Kawamoto and Hiroo Iwata
Bioconjugate Chemistry 2010 Volume 21(Issue 4) pp:792
Publication Date(Web):March 8, 2010
DOI:10.1021/bc900494x
Microencapsulation of islets with a semipermeable membrane, i.e., bioartificial pancreas, is a promising way to transplant islets without the need for immunosuppressive therapy for insulin-dependent diabetes mellitus (type I diabetes). However, materials composing a bioartificial pancreas are not ideal and might activate defense reactions against foreign materials. In this study, we propose an original method for microencapsulation of islets with living cells using an amphiphilic poly(ethylene glyocol)-conjugated phospholipid derivative (PEG-lipid) and DNA hybridization. PolyA and polyT were introduced onto the surfaces of the islets and HEK 293 cells, respectively, using amphiphilic PEG-lipid derivatives. PolyA20 modified HEK cells were immobilized onto the islet surface where polyT20-PEG-lipid was incorporated. The cells spread and proliferated on the islet surface, and the islet surface was completely encapsulated with a cell layer after culture. The encapsulated islets retained the ability to control insulin release in response to glucose concentration changes.
Co-reporter:Yuji Teramura and Hiroo Iwata
Soft Matter 2010 vol. 6(Issue 6) pp:1081-1091
Publication Date(Web):25 Jan 2010
DOI:10.1039/B913621E
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.
Co-reporter:Yuji Teramura, Hao Chen, Takuo Kawamoto, Hiroo Iwata
Biomaterials 2010 31(8) pp: 2229-2235
Publication Date(Web):
DOI:10.1016/j.biomaterials.2009.11.098
Co-reporter:Nguyen Minh Luan, Yuji Teramura, Hiroo Iwata
Biomaterials 2010 31(34) pp: 8847-8853
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.08.004
Co-reporter:Yusuke Arima, Masako Kawagoe, Masanori Furuta, Mitsuaki Toda, Hiroo Iwata
Biomaterials 2010 31(27) pp: 6926-6933
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.05.063
Co-reporter:Hiroko Miyazaki, Takeshi Maki, Koichi Kato and Hiroo Iwata
ACS Applied Materials & Interfaces 2009 Volume 1(Issue 1) pp:53
Publication Date(Web):December 31, 2008
DOI:10.1021/am800147x
Cell−cell interactions are considered to play critical roles in the development and physiology of most tissues. However, it is not straightforward to analyze cell−cell interactions with conventional cell culture in which cells are randomly distributed. To overcome this limitation, we employed here an antibody display to sort different cell types onto separate regions on a single substrate with microscale precision, taking advantage of the specific recognition of cell surface markers by surface-displayed antibodies. The results obtained with two sets of cell combinations, T cell/myelomonocytoid cell and neuron/astrocyte, demonstrate that antibody displays are feasible to establish a site-addressable coculture.Keywords: astrocyte; cell−cell interaction; coculture; multicellular; neuron; surface marker
Co-reporter:Yusuke Arima, Masako Kawagoe, Mitsuaki Toda and Hiroo Iwata
ACS Applied Materials & Interfaces 2009 Volume 1(Issue 10) pp:2400
Publication Date(Web):September 28, 2009
DOI:10.1021/am9005463
Hydrogels of polymers carrying surface hydroxyl groups strongly activate the complement system through the alternative pathway, although it has also been reported that solutions of polymers do not. To address these curious, inconsistent results, we examined the effect of polymer states, either immobilized on a surface or soluble in serum, on the complement activation using a surface plasmon resonance apparatus and enzyme-linked immunosorbent assay. We clearly showed that dextran- and poly(vinyl alcohol)-immobilized surfaces strongly activated the complement system but that soluble polymers could not, even when the amounts of the soluble polymers added to serum were 4−2000 times higher than those on the polymer-immobilized surfaces.Keywords: complement activation; dextran; poly(vinyl alcohol); protein adsorption
Co-reporter:Tadashi Nakaji-Hirabayashi, Koichi Kato and Hiroo Iwata
Bioconjugate Chemistry 2009 Volume 20(Issue 1) pp:102
Publication Date(Web):December 30, 2008
DOI:10.1021/bc800331t
To develop culture substrates for use in selective expansion of neural stem cells (NSCs), epidermal growth factor (EGF)-containing chimeric proteins were designed and synthesized by means of recombinant DNA technology. The chimeric proteins consisted of three components including an EGF domain, an α-helical oligopeptide, and a hexahistidine sequence. Two different α-helical oligopeptides were separately incorporated into chimeric proteins. Structural analyses by native gel electrophoresis and circular dichroism spectroscopy revealed that the heterodimer of these proteins was spontaneously formed through coiled-coil association of the α-helical oligopeptides. The monomeric and dimeric forms of these chimeric proteins were immobilized to the glass-based substrate via coordinate bonding between the hexahistidine and Ni(II) ions fixed on a substrate. The results of cell culture assays with NSCs showed that cells proliferated most rapidly and selectively on a substrate with the surface-anchored EGF dimer. The rate of cell proliferation on the surface with dimeric EGF was 1.3−2.0 times higher on the surfaces with monomeric EGF. In addition, the content of stem cells, determined 96 h after cell seeding, was highest on the surface with dimeric EGF (98%) among the surfaces studied (90−97% on surfaces with monomeric EGF). The observed growth rate and the stem cell content on the surface with EGF dimer were far beyond those in the standard neurosphere culture. The effect of surface-anchored dimeric EGF may be attributed to the enhanced dimerization of EGF-EGF receptor complexes leading to efficient signaling for mitogenic activity. We conclude that surface-anchoring of the EGF dimer provides an excellent substrate that allows the highly efficient expansion of NSCs.
Co-reporter:Makiko Hiraoka, Koichi Kato, Tadashi Nakaji-Hirabayashi and Hiroo Iwata
Bioconjugate Chemistry 2009 Volume 20(Issue 5) pp:976
Publication Date(Web):April 7, 2009
DOI:10.1021/bc9000068
To develop biomaterials that serve to improve the survival of neural cells transplanted into central nervous tissues, type I collagen-based hydrogels were prepared as a cell carrier. The hydrogels were modified with a laminin-derived peptide that is known to have an affinity for alpha3beta1 integrin, to transduce antiapoptotic signaling in embedded cells. For the modification of collagen, the peptide was fused to the N- or C-terminus, or both termini of a collagen-binding polypeptide domain by means of recombinant DNA technology. The chimeric proteins were characterized by polyacrylamide gel electrophoresis and circular dichroism spectroscopy, while binding of chimeric proteins to collagen-coated substrates was verified by surface plasmon resonance analysis under physiological conditions. Cell culture assays revealed that the adhesion of neurosphere-forming cells to collagen-coated polystyrene surfaces was significantly promoted by the incorporation of the chimeric proteins in a peptide-density dependent manner. The live/dead assays for cells cultured for 24 or 48 h in the hydrogels revealed that peptide incorporation improved the survival of cells embedded in collagen hydrogels. These results suggest that collagen hydrogel containing the laminin-derived peptide provides microenvironments suitable for the survival of neural cells.
Co-reporter:Yuji Teramura and Hiroo Iwata
Bioconjugate Chemistry 2008 Volume 19(Issue 7) pp:1389
Publication Date(Web):June 6, 2008
DOI:10.1021/bc800064t
Transplantation of islets of Langerhans (islets) is a promising technique for treating insulin-dependent diabetes mellitus (type I). One unresolved issue is early graft loss due to inflammation triggered by blood coagulating on the surface of islets after transplantation into the portal vein. Here, we describe a versatile method for modifying the surface of islets with an ultrathin membrane carrying the fibrinolytic enzyme urokinase or the anticoagulant heparin. The surface of islets was modified with a poly(ethylene glycol)−phospholipid conjugate bearing a biotin group (biotin-PEG-lipids, PEG MW: 5000). Biotin-PEG-lipids were anchored to the cell membranes of islets, and the PEG-lipid layer on the islets was further covered by streptavidin and biotin−bovine serum albumin conjugate using a layer-by-layer method. The surface was further activated with oxidized dextran. Urokinase was anchored to the islets through Schiff base formation. Heparin was anchored to the islets through polyion complex formation between anionic heparin and a cationic protamine coating on the islets. No practical islet volume increase was observed after surface modification, and the modifications did not impair insulin release in response to glucose stimulation. The anchored urokinase retained high fibrinolytic activity, which could help to improve graft survival by preventing thrombosis on the islet surface.
Co-reporter:Hiroko Miyazaki, Koichi Kato, Yuji Teramura and Hiroo Iwata
Bioconjugate Chemistry 2008 Volume 19(Issue 6) pp:1119
Publication Date(Web):May 14, 2008
DOI:10.1021/bc700470v
A chimeric protein consisting of a cell-adhesive peptide derived from a neural cell adhesion molecule and a collagen-binding domain was synthesized using recombinant DNA technology. Here, we demonstrate that the chimeric protein binds to type I collagen and promotes the adhesion and neurite extension of hippocampus neurons. These results suggest that the chimeric protein has potential to provide microenvironments for neurons to adhere and survive in collagen-based matrices for use in cell-based therapies for central nervous disorders.
Co-reporter:Tadashi Nakaji-Hirabayashi, Koichi Kato, Yusuke Arima and Hiroo Iwata
Bioconjugate Chemistry 2008 Volume 19(Issue 2) pp:516
Publication Date(Web):January 11, 2008
DOI:10.1021/bc700355t
Controlling the dynamics of growth factor signaling is a challenge in regenerative medicine for various tissues including the central nervous system. Here, we report on the development of the biomolecular system that facilitates sequential regulation of growth factor signals acting on neural stem/progenitor cells. Recombinant technology was employed to synthesize the multifunctional chimeric protein that contained multiple domains, including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), globular capping domain, thrombin-cleavable sequence, and substrate-binding domain with affinity for Ni(II) ions. The chimeric protein is expected to expose CNTF upon elimination of the capping domain by digestion with endogenous thrombin in vivo. When the multifunctional chimeric protein was immobilized onto a substrate through the coordination of the substrate-binding domain with surface-immobilized Ni(II) ions, the substrate served to proliferate neural stem cells, maintaining the population of undifferentiated cells at 85%. This effect is primarily due to the activity of EGF, while CNTF activity is temporally veiled with the capping domain. Upon digesting the thrombin-cleavable sequence to remove the capping domain, the activity of CNTF emerged to induce differentiation of astrocytes in situ from the proliferated neural stem cells. The fraction of differentiated astrocytes reached 68% of total cells. These results demonstrate the feasibility of the system for controlling the dynamics of growth factor signals.
Co-reporter:Hiroyuki Fujimoto, Koichi Kato, Hiroo Iwata
Analytical Biochemistry 2008 Volume 374(Issue 2) pp:417-422
Publication Date(Web):15 March 2008
DOI:10.1016/j.ab.2007.12.029
Co-reporter:Carlos A. Agudelo, Hiroo Iwata
Biomaterials 2008 Volume 29(Issue 9) pp:1167-1176
Publication Date(Web):March 2008
DOI:10.1016/j.biomaterials.2007.11.027
Bioartificial pancreas in which islets of Langerhans (islets) are enclosed in a semipermeable membrane is one of the approaches to treat insulin-dependent diabetic patients. Although there are advantages in this method, one of the issues that still remains is the long-term storage of tissue engineering devices before transplantation. One of the possible routes to address this is through cryopreservation. In this study, a freezing solution, 2 m DMSO in RPMI-1640, a conventional vitrification solution, VS55, and the newly developed vitrification solution KYO-1 were examined to cryopreserve microencapsulated islets in agarose hydrogel. The insulin release ability, morphology of islets, and physico-chemical properties of the agarose gel membrane were examined after a cryopreservation and thawing process. Frozen and vitrified (by KYO-1) groups showed a similar insulin secretion. Frozen groups by 2 m DMSO, however, showed destruction of agarose capsules and some islets were out of the capsule. When KYO-1 was used, islets still maintained the ability to release insulin in response to glucose stimulation, and agarose capsule showed morphological integrity, and mechanical properties. In conclusion, vitrification using KYO-1 which is composed of 5.38 m ethylene glycol, 2 m DMSO, 0.1 m PEG 1000 and 0.00175 m PVP K10 in EuroCollins, is a suitable method for cryopreservation of microencapsulated islets.
Co-reporter:S. Koda, Y. Inoue and H. Iwata
Langmuir 2008 Volume 24(Issue 23) pp:13525-13531
Publication Date(Web):November 7, 2008
DOI:10.1021/la8021358
Gene transfection into adherent cells from plasmid DNA (pDNA)-arrayed substrates known as gene transfection arrays appears to be a promising tool for the high-throughput analysis of gene functions and protein−protein interaction networks. We tested the ability of electric pulse-stimulated gene transfection from a substrate to overcome low expression efficiency and cross contamination between spots on arrays. We prepared the electrodes used for electric pulse-stimulated gene transfection by sequentially loading a gold thin layer, a self-assembled monolayer of a carboxylic acid-terminated alkanethiol (COOH-SAM), and poly(amidoamine) (PAMAM) dendrimers, either through electrostatic interactions or by covalent linkage to COOH-SAM and then to pDNA. When dendrimers were loaded onto the electrode using electrostatic interactions, the gene-expression efficiency of adherent cells increased as the generation numbers of the dendrimers that we used increased. Gene expression was rarely observed in adherent cells when dendrimers were covalently immobilized onto the electrode. Additionally, we successfully demonstrated site-specific gene transfer using a dendrimer-array electrode with no cross contamination between spots on the electrode.
Co-reporter:Tadashi Nakaji-Hirabayashi, Koichi Kato and Hiroo Iwata
Biomacromolecules 2008 Volume 9(Issue 5) pp:
Publication Date(Web):April 23, 2008
DOI:10.1021/bm701423d
The poor survival of neural stem/progenitor cells following transplantation into the brain is the major problem limiting the effect of cell-based therapy for Parkinson’s disease. To overcome this problem, we are involved in designing keratin-based hydrogels that serve as physical barriers to prevent the infiltration of inflammatory cells. Another feature of the hydrogels is to contain a polypeptide that promotes integrin-mediated cell adhesion. To construct such hydrogels, a chimeric protein consisting of an α-helical polypeptide and a globular domain derived from laminin was synthesized by means of recombinant DNA technology and coassembled with extracted keratins that form hydrogels through intermolecular coiled-coil association of α-helical segments. It was found that neurosphere-forming cells specifically adhered to the keratin-based composite hydrogel and actively proliferated at a high survival rate. These results suggested that the composite hydrogel provides microenvironments suitable for the survival and proliferation of neural progenitor cells.
Co-reporter:Hiroyuki Fujimoto;Koichi Kato
Analytical and Bioanalytical Chemistry 2008 Volume 392( Issue 7-8) pp:
Publication Date(Web):2008 December
DOI:10.1007/s00216-008-2423-z
This paper describes the fabrication of microarrays that enable the parallel electroporation of small interfering RNAs (siRNAs) into mammalian cells. To optimize the conditions of microarray preparation and electric pulsing, a self-assembled monolayer was formed on a gold electrode, and a cationic polymer was adsorbed by the entire surface of the monolayer. siRNA was then adsorbed by the cationically modified electrode through electrostatic interactions. Human embryonic kidney cells stably transformed with the expression construct of green fluorescent protein (GFP) were used to examine the electric pulse-triggered transfer of GFP-specific siRNA. A single electric pulse was applied to the cells cultured on the electrode at a field strength of 240 V cm−1. The expression of GFP was significantly suppressed in a sequence-specific manner two days after pulsing. Microscopic observation and flow-cytometric analysis revealed that the expression of GFP was attenuated in the majority of cells in a loading-dependent manner. Moreover, the effect of siRNA could be temporally controlled by changing the culture periods before pulsing. When a micropatterned self-assembled monolayer was used as a platform for loading siRNA in an array format, gene silencing was spatially restricted to the regions where specific siRNA was loaded. From these results, we conclude that array-based electroporation provides an excellent means of individual transfer of siRNAs into mammalian cells for high-throughput gene function studies.
Co-reporter:Ellyana Njatawidjaja
Analytical and Bioanalytical Chemistry 2008 Volume 392( Issue 3) pp:405-408
Publication Date(Web):2008 October
DOI:10.1007/s00216-008-2281-8
The transfectional microarray is a promising tool to conduct high-throughput analysis of gene function. In this study, a miniaturized multiwell plate was prepared by applying a silicone rubber sheet with holes on a gold electrode. The combination of electroporation and a miniaturized multiwell plate successfully achieved spatially controlled gene delivery with absence of cross-contamination between genes. Furthermore, we found that gene delivery efficiency was not dependent on conditions such as plasmid loading, electric field strength, and pulse duration.
Co-reporter:Satoshi Fujita;Yutaka Morita
Analytical and Bioanalytical Chemistry 2008 Volume 391( Issue 8) pp:2753-2758
Publication Date(Web):2008 August
DOI:10.1007/s00216-008-1921-3
Conventional assays for hematopoietic progenitor cells (HPCs) require long-term culturing, a labor-intensive procedure, and technique proficiency. We aimed to develop a high-throughput method to determine frequency of quiescent primitive HPCs by a combination of the micro-multiwell plate and 5-fluorouracil (5-FU) treatment. The micro-multiwell plate was made of a silicone sheet with a 6 × 6 array of 1-mm diameter holes and a glass substrate. To enrich primitive HPCs in a CD34 population, CD34 cells and stromal cells were applied to micro-multiwells and cultured in the presence of 5-FU for 2 days. The quiescent primitive HPCs that survived after 5-FU treatment were then expanded with cytokines in the absence of 5-FU for a further 10 days. After culturing, cells were immunostained and the number of primitive HPCs in inoculated CD34 cells was estimated from fluorescent intensity for each well under a stereoscopic fluorescent microscope. The frequencies of primitive HPCs correlated well with frequencies of cobblestone area-forming cells for two CD34 cell lots. Our method allows high-throughput screening for primitive HPCs in CD34 cells.
Co-reporter:Yusuke Arima and Hiroo Iwata
Journal of Materials Chemistry A 2007 vol. 17(Issue 38) pp:4079-4087
Publication Date(Web):10 Aug 2007
DOI:10.1039/B708099A
We investigated initial cell adhesion on self-assembled monolayers (SAMs) of alkanethiols carrying different functional groups including methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), and amine (NH2). The combination of a surface plasmon resonance (SPR) instrument and a total internal reflection fluorescence microscope (TIRFM) allowed us to examine the kinetics of protein adsorption and correlating cell adhesion. Upon exposure of the SAM surface to a serum-containing medium, serum proteins rapidly adsorbed, and cells subsequently approached the surface. Adhesion of human umbilical vein endothelial cells (HUVECs) was greatly affected by surface functional groups; HUVECs adhered well to COOH– and NH2–SAMs, whereas poorly to CH3– and OH–SAMs. The amount of adsorbed protein from the serum-containing medium varied slightly with the terminal groups of the SAMs. On COOH– and NH2–SAMs, HUVECs adhered to bovine serum albumin (BSA)-preadsorbed surfaces with a few minutes delay, suggesting that displacement of preadsorbed BSA with cell-adhesive proteins, such as fibronectin or vitronectin, supports cell adhesion to these surfaces. Since the concentration of cell-adhesive proteins is much less than that of non-adhesive proteins such as BSA, displacement of adsorbed proteins with cell-adhesive proteins plays an important role in initial cell adhesion.
Co-reporter:Koichi Kato, Hideki Sato and Hiroo Iwata
Bioconjugate Chemistry 2007 Volume 18(Issue 6) pp:2137
Publication Date(Web):September 26, 2007
DOI:10.1021/bc700259g
In an attempt to develop collagen-growth factor composites for use in tissue engineering, chimeric proteins consisting of epidermal growth factor and collagen binding domains derived from von Willebrand factor or fibronectin were synthesized by means of recombinant technology. These chimeric proteins were bound to type I collagen fibrils, and the ultrastructures of composites were analyzed by transmission electron microscopy combined with the gold nanoparticle labeling technique. The results of the ultrastructural study revealed that chimeric proteins were densely assembled on collagen fibrils through the specific recognition of binding sites, producing the ordered array of chimeric proteins.
Co-reporter:Hao Chen, Hideki Sato, Takahiko Totani, Hiroo Iwata
Analytical Biochemistry 2007 Volume 366(Issue 2) pp:137-143
Publication Date(Web):15 July 2007
DOI:10.1016/j.ab.2007.04.008
Embryonic stem (ES) cells hold promise as a source for cell transplantation treatment of diseases such as type I diabetes. Further, cells releasing bioactive substances from ES cell progeny may be concentrated and purified for clinical applications. Although ES cell lines that express reporter genes have been established to isolate cells releasing bioactive substances, other difficulties must be overcome before these genetically modified cells can be used for gene therapy in human patients. Fluorescence- or magnetic-activated cell sorters are commonly used to isolate specific cells using antibodies against cell surface antigens. However, for some cells, such as insulin-producing beta cells, specific surface antigens have not yet been identified. In this study, we developed a simple and efficient method to identify and purify insulin- and alpha-fetoprotein-producing cells. A nitrocellulose membrane treated with anti-insulin or anti-alpha–fetoprotein antibodies was placed on a cell layer to trap insulin or alpha-fetoprotein released from the cells. The location of specific substance-producing cells was identified by immunostaining the membrane. The insulin-releasing cells were selectively collected from the culture dish using a cloning ring and transferred to another culture plate.
Co-reporter:Hironori Yamazoe;Kenta Moriyasu;Kenta Moriyasu;Hironori Yamazoe
Journal of Biomedical Materials Research Part A 2006 Volume 77A(Issue 1) pp:136-147
Publication Date(Web):3 JAN 2006
DOI:10.1002/jbm.a.30576
Cell transplantation therapy using dopaminergic neurons derived from embryonic stem (ES) cells for the treatment of Parkinson's disease has been proposed as one of the major applications for stem cell-based therapy. However, the low collection efficiency of neurons from a culture dish and the rejection of cells after transplantation are expected to limit their future clinical applications. To overcome these problems, we examined the induction of neurogenis of ES cells under free-floating conditions and microencapsulation of the obtained cell aggregates into an agarose hydrogel. Cell aggregates from ES cells were cultured in various media under the free-floating condition. Immunohistochemical staining for tyrosine hydroxylase (TH) and RT-PCR analyses for TH and Nurr1 showed that dopaminergic neurons were induced in ES cell aggregates cultured in a 1:2 mixture of conditioned medium of PA6 stromal cells and Glasgow minimum essential medium (GMEM) after 16 days in culture. The cell aggregates could be collected and were encased within agarose microcapsules without loss of dopaminergic neurons. The cell aggregates with/without microencapsulation were maintained in CM/GMEM for an additional period. KCl stimulation assays were done at day 23, 30, 37, 44, 51, and 58 to examine dopamine release. Dopamine release abilities were well maintained during 58 days of observation. Amounts of dopamine release from encapsulated cell aggregates were slightly higher than those of unencapsulated cell aggregates from day 16 to 58. Although efficacy for immunoisolation of the agarose microcapsules still remains for future in vivo studies, microencapsulation did not adversely affect viability and functions of the dopamine releasing ES cell progeny. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Co-reporter:Yuji Teramura, Yusuke Arima, Hiroo Iwata
Analytical Biochemistry 2006 Volume 357(Issue 2) pp:208-215
Publication Date(Web):15 October 2006
DOI:10.1016/j.ab.2006.07.032
In this study, we developed a surface plasmon resonance (SPR)-based sensing method with high sensitivity for the detection of brain natriuretic peptide (BNP), the level of which is a useful indicator of heart failure. In our system, BNP was detected by a sandwich-type immunoassay using two kinds of monoclonal antibodies: primary and secondary. The SPR signal was amplified by using streptavidin-conjugated nanobeads approximately 50 nm in diameter that specifically accumulated on the secondary antibody via biotin–avidin bonding. By this method, the SPR signals were highly intensified, and thus picogram levels (pg/ml) of BNP could be detected. The streptavidin nanobead amplification method realizes detection of very low levels of BNP, as is necessary for the clinical diagnosis. It is also expected that our streptavidin nanobead amplification method can be applicable to the detection of other hormones and tumor markers that are present in only low concentrations within the human body.
Co-reporter:Suguru Miura, Yuji Teramura, Hiroo Iwata
Biomaterials 2006 Volume 27(Issue 34) pp:5828-5835
Publication Date(Web):December 2006
DOI:10.1016/j.biomaterials.2006.07.039
The microencapsulation of islets of Langerhans (islets) has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. However, thinner membranes are desired, because the increased total volume of the implant led to limited transplantation sites. Here, we propose a novel method for microencapsulation by polyion complex membrane formation on islets. Amino group-terminated poly(ethylene glycol)-conjugated phospholipids (PEG-lipids, Mw: 5000) spontaneously formed a thin layer on cells existing in the outer layer of islets when they were added to islet suspension. This layer-by-layer membrane could be further formed on the PEG-lipid layer through polyion complex formation between amino groups at the end of PEG chains, sodium alginate and poly(l-lysine). Islets could be microencapsulated by this method without volume increase. Encapsulation of the islet surface with PEG-lipids and polyion complex membranes did not impair the insulin release function in response to glucose stimulation. Our method is promising to encapsulate islets without affecting cell viability or increasing volume.
Co-reporter:Hironori Yamazoe, Yoshinobu Murakami, Kenji Mizuseki, Yoshiki Sasai, Hiroo Iwata
Biomaterials 2005 Volume 26(Issue 28) pp:5746-5754
Publication Date(Web):October 2005
DOI:10.1016/j.biomaterials.2005.02.021
Embryonic stem (ES) cells have the ability to replicate themselves and differentiate into various mature cells. Recently, dopaminergic neurons were efficiently induced from ES cells using mouse stromal cells (PA6 cells) as a feeder cell layer. This simple procedure seems to be very efficient to obtain dopamine-releasing cells for future clinical cell transplantation treatment of Parkinson's disease. In this study, we prepared stock solutions containing neural inducing factors (NIFs) by washing PA6 cells with phosphate-buffered saline containing heparin. ES cells grew successfully in culture media supplemented with 33 v/v% NIFs stock solution, and the rate of neural differentiation of ES cell progeny increased with increasing heparin concentration in the culture media. In addition, NIFs-immobilized surfaces were prepared by exposing polyethyleneimine-modified surfaces to NIFs stock solutions. The NIFs-immobilized culture dish effectively supported cell growth as the culture medium supplemented with NIFs stock did, but its induction effect to dopaminergic neurons from ES cells was much smaller than free NIFs. NIFs stock solutions have two different activities. One can stimulate cell growth and the other induces differentiation of ES cells to the neural fate when heparin existed. The former factors were effectively immobilized on the culture dish, but those that induce differentiation may not be. Further optimization is required.
Co-reporter:Yoshinobu Murakami;Yoshito Ikada;Isao Hirata;Takayuki Kitazawa;Hajime Kitamura;Mitsuaki Toda;Yasunori Hioki;Etsuko Kitano
Journal of Biomedical Materials Research Part A 2003 Volume 66A(Issue 3) pp:669-676
Publication Date(Web):5 AUG 2003
DOI:10.1002/jbm.a.10067
Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 669–676, 2003
Co-reporter:Eiji Itoh;Koji Yamauchi;Takashige Oka;Yoshio Yamaoka;Shojiro Matsuda;Yoshito Ikada
Journal of Biomedical Materials Research Part A 2000 Volume 53(Issue 6) pp:640-645
Publication Date(Web):20 NOV 2000
DOI:10.1002/1097-4636(2000)53:6<640::AID-JBM4>3.0.CO;2-L
Staple-line reinforcement buttresses made of bovine pericardium (BP), expanded polytetrafluoroethylene (ePTFE), and so on have been shown to be effective in preventing air leaks after stapled lung volume reduction operations, and some of them have been clinically utilized. However, each buttress suffers at least one disadvantage such as risk of viral infection and chronic inflammation. A new buttress was made using a poly(L-lactic acid-co-ϵ-caprolactone) film (L/C film) and its effectiveness as a staple-line reinforcement was examined by performing lung volume reduction operation on a canine model. Soft tissue responses to the buttress were compared with those to the BP strip and the absorbable behavior was studied. The L/C film buttress was flexible and thin enough to easily cut. Death of dogs, infection, acute and prolonged air leaks, and any complications related to its use were not observed. The tissue responses to the film were more mild and favorable than those to BP. The L/C film was absorbed after the staple line was covered by a connective tissue. The results described above suggest that the buttress made of an L/C film is a promising staple-line reinforcement material. © 2000 John Wiley & Sons, Inc. Biomed Mater Res (Appl Biomater) 53: 640–645, 2000
Co-reporter:Isao Hirata, Yoko Morimoto, Yoshinobu Murakami, Hiroo Iwata, Etsuko Kitano, Hajime Kitamura, Yoshito Ikada
Colloids and Surfaces B: Biointerfaces 2000 Volume 18(3–4) pp:285-292
Publication Date(Web):October 2000
DOI:10.1016/S0927-7765(99)00154-X
It has been accepted that covalent immobilization of C3b on artificial materials is the most important step to initiate the complement activation. However, there are few studies that have directly demonstrated covalent immobilization of C3b on artificial surfaces. In this study, model thin layers were prepared by the self-assembled monolayer method to produce a surface covered with hydroxyl or methyl groups using mercaptododecane (CH3-SAM) and mercaptoundecanol (OH-SAM). Interactions of the complement system with the model surfaces were studied using a surface plasmon resonance instrument. The OH-SAM immobilized C3b, resulting in activating of the complement system through the alternative pathway in Veronal-buffered saline, but this surface did not activate the classical pathway. However, the OH-SAM could not activate the alternative pathway in Veronal-buffered saline containing 10 mM EGTA and 2 mM MgCl2 that is believed not to interfere with the activation of the alternative pathway. The hydrophobic CH3-SAM surface could not activate the classical pathway, but activated the alternative pathway, although the extent was small.
Co-reporter:Hisayuki Oowaki, Shojiro Matsuda, Nobuyuki Sakai, Takayuki Ohta, Hiroo Iwata, Akiyo Sadato, Waro Taki, Nobuo Hashimoto, Yoshito Ikada
Biomaterials 2000 Volume 21(Issue 10) pp:1039-1046
Publication Date(Web):May 2000
DOI:10.1016/S0142-9612(99)00278-1
Endovascular neurosurgery is now becoming available as one of strategies for the treatment of cerebro-spinal arterio-venous malformations and aneurysms. For this treatment, a microcatheter is advanced into or close to a lesion and then an embolic material is administered through it to obliterate the lesion. N-butyl-2-cyanoacrylate (NBCA) has preferentially been used as an embolic material in Europe and America. However, its exceptionally strong adhesive force sometimes causes adhesion between the tip of the microcatheter and the artery. In this study, a new non-adhesive cyanoacrylate, isostearyl-2-cyanoacrylate (ISCA), was developed. It carries a long hydrophobic side isostearyl group with lower reactivity and adhesion than other cyanoacrylates. Its polymerization rate is, however, too low to obliterate a vascular lesion with a rapid blood flow. To increase the polymerization rate, ISCA was mixed with NBCA. As a result, the adhesive force of the mixture became extremely low, compared with that of NBCA. The viscosity of the mixture was low enough to allow its’ use as an embolic material. Tissue reactions against the mixture was milder than those against NBCA. Radio-angiography became possible by mixing further with Lipiodol. The evaluation of this new embolic material with a rabbit renal artery showed that the obliteration effect of the mixture of ISCA and NBCA was excellent to use as an embolic material for clinical applications.
Co-reporter:Yusuke Arima, Mitsuaki Toda, Hiroo Iwata
Advanced Drug Delivery Reviews (16 September 2011) Volume 63(Issue 12) pp:988-999
Publication Date(Web):16 September 2011
DOI:10.1016/j.addr.2011.06.018
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.Download high-res image (321KB)Download full-size image
Co-reporter:Yuji Teramura, Hiroo Iwata
Advanced Drug Delivery Reviews (15 June 2010) Volume 62(Issues 7–8) pp:827-840
Publication Date(Web):15 June 2010
DOI:10.1016/j.addr.2010.01.005
Type 1 diabetes has been successfully treated by transplanting islets of Langerhans (islets), endocrine tissue releasing insulin. Serious issues, however, still remain. The administration of immunosuppressive drugs is required to prolong graft functioning; however, side effects of their long-term use on recipients are not fully understood, and cell transplantation therapy without the use of immunosuppressive drugs is desired. To resolve these issues, the encapsulation of isles with a semi-permeable membrane, or bioartificial pancreas, has been attempted. Many groups have reported that it functions very well in small animal models. Few of the bioartificial pancreases, however, were applied to human patients and their clinical outcome was not clear. In this review, we address obstacles and overview new techniques to overcome these issues, such as conformal coating and islet enclosure with cells.
Co-reporter:Yuji Teramura, Hiroo Iwata
Analytical Biochemistry (15 June 2007) Volume 365(Issue 2) pp:201-207
Publication Date(Web):15 June 2007
DOI:10.1016/j.ab.2007.03.022
In this study, we attempted to develop a surface plasmon resonance (SPR)-based immunoassay sensor to detect α-fetoprotein (AFP) in human plasma at the nanogram level, as is required for clinical diagnosis of hepatocellular tumors. A self-assembled monolayer (SAM) surface of tri(ethylene glycol) (TEG) and carboxyl group-terminated hexa(ethylene glycol) (HEG) was employed to suppress the nonspecific adsorption of plasma components onto the sensor surface. AFP was detected by a sandwich-type immunoassay using two kinds of antibodies, primary and secondary, in this system. The SPR signal shift was further enhanced by applying an antibody (polyclonal) against the second antibody. With this method, the SPR signals were highly intensified, and so nanogram levels (ng/ml) of AFP could be easily detected with a high signal/noise ratio, as is necessary for clinical diagnosis. It is expected that our SPR-based immunoassay method can also be applicable to the detection of several other tumor markers that are present in low concentrations in human blood.
Co-reporter:Yusuke Arima and Hiroo Iwata
Journal of Materials Chemistry A 2007 - vol. 17(Issue 38) pp:NaN4087-4087
Publication Date(Web):2007/08/10
DOI:10.1039/B708099A
We investigated initial cell adhesion on self-assembled monolayers (SAMs) of alkanethiols carrying different functional groups including methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), and amine (NH2). The combination of a surface plasmon resonance (SPR) instrument and a total internal reflection fluorescence microscope (TIRFM) allowed us to examine the kinetics of protein adsorption and correlating cell adhesion. Upon exposure of the SAM surface to a serum-containing medium, serum proteins rapidly adsorbed, and cells subsequently approached the surface. Adhesion of human umbilical vein endothelial cells (HUVECs) was greatly affected by surface functional groups; HUVECs adhered well to COOH– and NH2–SAMs, whereas poorly to CH3– and OH–SAMs. The amount of adsorbed protein from the serum-containing medium varied slightly with the terminal groups of the SAMs. On COOH– and NH2–SAMs, HUVECs adhered to bovine serum albumin (BSA)-preadsorbed surfaces with a few minutes delay, suggesting that displacement of preadsorbed BSA with cell-adhesive proteins, such as fibronectin or vitronectin, supports cell adhesion to these surfaces. Since the concentration of cell-adhesive proteins is much less than that of non-adhesive proteins such as BSA, displacement of adsorbed proteins with cell-adhesive proteins plays an important role in initial cell adhesion.
Co-reporter:Naohiro Takemoto, Yuji Teramura and Hiroo Iwata
Biomaterials Science (2013-Present) 2013 - vol. 1(Issue 3) pp:NaN321-321
Publication Date(Web):2012/11/28
DOI:10.1039/C2BM00048B
Sertoli cells play a crucial role in creating the immunoprivileged environment of the testis. We examined the survival of islets of Langerhans after co-transplantation with Sertoli cells. Sertoli cells near islets should protect the graft from rejection. In this study, conjugates of single stranded oligonucleotides, poly(ethylene glycol) and phospholipids (ssDNA-PEG-DPPE) were used to immobilize Sertoli cells on islets. The 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of deoxythymidylic acid (oligo(dT)20) were presented as ssDNAs on the surfaces of Sertoli cells and islets, respectively, through the hydrophobic interaction between a lipid unit of the conjugates and the cell membrane. The Sertoli cells were immobilized on the islets through hybridization between oligo(dA)20 and oligo(dT)20. When Sertoli cell-immobilized islets were infused into the liver of mice through the portal vein, the Sertoli cells remained around the islets.
![Poly[oxy[(1S)-1-methyl-2-oxo-1,2-ethanediyl]]](http://img.cochemist.com/ccimg/26200/26161-42-2.png)
![Poly[oxy[(1S)-1-methyl-2-oxo-1,2-ethanediyl]]](http://img.cochemist.com/ccimg/26200/26161-42-2_b.png)
