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Biomedical subjects

Teruo Okano

Publications and source records attributed to Teruo Okano.

At least 73 records · Page 4Linked to original sources

Regulation of sialyl-Lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line.

Sialyl-Lewis x epitopes and MUC5AC protein are known to be overexpressed in mucins secreted by patients suffering from various respiratory diseases. To investigate the mechanisms by which airway inflammatory agents mediate the expression of sialyl-Lewis x epitopes and MUC5AC mucin, we examined the effects of tumor necrosis factor (TNF)-alpha and epidermal growth factor (EGF) in the human lung carcinoma cell line, NCI-H292. Basal expression levels of hST3GalIV, FUT3 and C2/4GnT mRNA, involved in the biosynthesis of sialyl-Lewis x, were higher than those of other glycosyltransferases in NCI-H292 cells. TNF-alpha induced expression of hST3GalIV, FUT3, C2/4GnT and MUC5AC mRNAs in NCI-H292 cells. When cells were pretreated with U73122, a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor, the expression of these glycosyltransferase mRNAs was suppressed. Treating cells with EGF induced the down-regulation of these glycosyltransferase mRNAs and sialyl-Lewis x epitopes, while inducing an increase in expression of MUC5AC mRNA. These EGF-mediated effects on the glycosyltransferase and MUC5AC mRNAs were blocked when cells were first exposed to AG1478, an EGF receptor tyrosine kinase inhibitor. These findings suggest that the expression of sialyl-Lewis x epitopes, which is regulated separately from the expression of MUC5AC protein, may be controlled through pathways such as the EGF receptor tyrosine kinase and PI-PLC signaling cascades in NCI-H292 cells.

Cell Line, Tumor↗

The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets.

Heterotypic cell interactions are critical to achieve and maintain specific functions in many tissues and organs. We have focused on patterned structure surfaces to enable co-culture of heterotypic cells and recovery of patterned co-cultured cell sheets for applications in tissue engineering. Thermoresponsive polymers exhibiting different transition temperatures in water comprise both poly(N-isopropylacrylamide) (PIPAAm) and n-butyl methacrylate (BMA) co-grafted as side chains to PIPAAm main chains. These copolymers were surface-grafted in patterns to obtain patterned dual thermoresponsive cell culture surfaces using electron beam polymerisation method and porous metal masks. On patterned surfaces, site-selective adhesion on and growth of rat primary hepatocytes (HCs) and bovine carotid endothelial cells (ECs) allowed patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by culture temperature as the sole variable. At 27 degrees C, seeded HCs adhered exclusively onto hydrophobic, dehydrated P(IPAAm-BMA) co-grafted domains (1-mm laser dot), but not onto neighbouring hydrated PIPAAm domains. Sequentially seeded ECs then adhered exclusively to hydrophobised PIPAAm domains upon increasing culture temperature to 37 degrees C, achieving patterned co-cultures. Reducing culture temperature to 20 degrees C promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets with heterotypic cell interactions. Recovered co-cultured cell sheets can be manipulated, moved and sandwiched with other structures, providing new useful constructs both for basic cell biology research and preparation of tissue-mimicking multi-layer materials through overlaying co-cultured cell sheets.

Acrylic Resins↗

Influence of insulin immobilization to thermoresponsive culture surfaces on cell proliferation and thermally induced cell detachment.

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.

Acrylamides↗

Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation.

Most of the surface patterning methods currently applied are based on lithography techniques and microfabrication onto silicon or glass substrates. Here we report a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilising both electron beam (EB) polymerisation and local laser ablation techniques for microfabrication. Polyacrylamide was grafted onto tissue culture polystyrene (TCPS) dishes using EB irradiation. Water contact angles for these PAAm-grafted TCPS surfaces were less than 10 degrees (costheta = 0.99) with PAAm grafted amounts of 1.6 microg/cm(2) as determined by ATR/FT-IR. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces composed of hydrophilic PAAm and hydrophobic basal polystyrene layers. Bovine carotid artery endothelial cells adhered only to the ablated domains after pretreatment of the patterned surfaces with 15 microg/mL fibronectin at 37 degrees C. The ablated domain sizes significantly influenced the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without loss of pattern fidelity, indicating that more durable cell arrays can be obtained compared to those prepared by self-assembled monolayers of alkanethiols, as described in previous reports. The surface fabrication techniques presented here can be utilised for the preparation of cell-based biosensors as well as tissue engineering constructs.

Acrylic Resins↗

Cell sheet engineering: recreating tissues without biodegradable scaffolds.

While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.

Animals↗

Repair of impaired myocardium by means of implantation of engineered autologous myoblast sheets.

OBJECTIVES: Autologous skeletal myoblast cell transplantation by means of the injection method is subject to the loss of intercellular communication, extracellular matrix, and cell numbers. We hypothesize that the implantation of skeletal myoblast cell sheets might be more advantageous in repairing the impaired heart by providing uniform and stable cell delivery with less cell loss and without disrupting the cell-cell microenvironment. METHODS: Left anterior descending coronary artery-ligated Lewis rat hearts (2 weeks, total n = 173) received 1 x 10(7) autologous skeletal myoblasts by means of cell transplantation either through myoblast injection or implantation of 2 monolayer-constructed myoblast sheets (5 x 10(6) cells per sheet) or through medium injection. Myoblast sheets were constructed with temperature-responsive, polymer-grafted cell-culture dishes, which release the confluent cells from the dish surface at less than 20 degrees C. RESULTS: Echocardiographic results indicated higher improvement of cardiac performance in the myoblast sheet group than among the other groups until 8 weeks after cell transplantation. Histologic comparison revealed greater cellularity and abundant widespread neocapillaries within the noticeable uniform thickened wall in myoblast sheet group hearts only. Fibrosis was substantially reduced with skeletal myoblast sheet implantation compared with skeletal myoblast cell injection. Obviously higher numbers of hematopoietic stem cells (c-kit, stem cell antigen 1, and CD34) were observed in the myoblast sheet group infarct heart region. Reverse transcription-polymerase chain reaction results showed expression of stromal-derived factor 1, hepatocyte growth factor, and vascular endothelial growth factor as follows: myoblast sheets > myoblast injection > control. CONCLUSIONS: Myoblast sheets repaired the impaired myocardium, reduced fibrosis, and prevented remodeling in association with recruitment of hematopoietic stem cells through the release of stromal-derived factor 1 and other growth factors. Our experiment indicates a therapy for patients with severe heart failure.

Animals↗

Novel and simple method for isolating autologous mesothelial cells from the tunica vaginalis.

OBJECTIVE: To report the development of a new method of isolating autologous mesothelial cells from the tunica vaginalis that are easily obtained and generally free from the effects of abdominal cancer, and to investigate whether transplanting these mesothelial cells is effective in preventing postoperative adhesions. MATERIALS AND METHODS: The tunica vaginalis was resected from male Lewis rats, and mesothelial cells were collected by enzymatic disaggregation. To investigate the efficacy of mesothelial cells in preventing adhesion, harvested cells were transplanted into a rat intestinal hernia adhesion model. RESULTS: Cells isolated from the tunica vaginalis were homogenous, polygonal when confluent, expressed cytokeratin and vimentin, and the cell surface was covered with microvilli, which is the characteristic appearance of endogenous mesothelial cells. The transplantation of autologous mesothelial cell sheets reduced peritoneal adhesion. CONCLUSION: We developed a new method of obtaining autologous mesothelial cells from the tunica vaginalis. These cells may provide a valuable option for treating patients at risk of postoperative adhesions.

Animals↗

A new method for manufacturing cardiac cell sheets using fibrin-coated dishes and its electrophysiological studies by optical mapping.

We developed a novel simple method for making functional myocardial cell sheets that may be used as transplants. Polymerized human fibrin-coated dishes were prepared with fibrinogen monomers mixed with thrombin. Neonatal rat cardiomyocytes cultured on these dishes formed myocardial cell sheets within 4 days. These cell sheets were easily dissociated intact from the polymerized fibrin layer, because the fibrin had been digested by intrinsic protease. Two overlaid myocardial cell sheets exhibited synchronized spontaneous beating and captured artificial pacing. Optical mapping confirmed that the conduction of the action potential between two partially overlaid myocardial cell sheets was established, and the action potential propagated across the junction without any delay. Transplanted three-layered myocardial cell sheets exhibited strong spontaneous beating and showed well-differentiated striations and an increase in cell size. This simple method of cell sheet engineering may also be applicable for various other cell types.

Animals↗

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs.

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.

Adjuvants, Immunologic↗

Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface.

PURPOSE: Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces. METHODS: Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined. RESULTS: All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium. CONCLUSIONS: Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.

Animals↗

[Drug delivery systems using nano-sized drug carriers].

Nanotechnology has attracted great attention all over the world in recent several years and has led to the establishment of the novel technical field of "nanomedicine" through collaboration with advanced medical technology. Particularly, site-specific drug targeting using particle drug carrier systems has made substantial progress and been actively developed. This review explains the essential factors (size and chemical character) of drug carriers to allow long circulation in the bloodstream avoiding the reticuloendothelial system, and shows the present status and future perspective of several types of nano-carrier systems (water-soluble polymer, liposome and polymeric micelle). We also introduce the novel concept of multi-targeting system (combination of two or more targeting methodologies) for ideal drug therapies.

Acrylic Resins↗

Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.

BACKGROUND: Ocular trauma or disease may lead to severe corneal opacification and, consequently, severe loss of vision as a result of complete loss of corneal epithelial stem cells. Transplantation of autologous corneal stem-cell sources is an alternative to allograft transplantation and does not require immunosuppression, but it is not possible in many cases in which bilateral disease produces total corneal stem-cell deficiency in both eyes. We studied the use of autologous oral mucosal epithelial cells as a source of cells for the reconstruction of the corneal surface. METHODS: We harvested 3-by-3-mm specimens of oral mucosal tissue from four patients with bilateral total corneal stem-cell deficiencies. Tissue-engineered epithelial-cell sheets were fabricated ex vivo by culturing harvested cells for two weeks on temperature-responsive cell-culture surfaces with 3T3 feeder cells that had been treated with mitomycin C. After conjunctival fibrovascular tissue had been surgically removed from the ocular surface, sheets of cultured autologous cells that had been harvested with a simple reduced-temperature treatment were transplanted directly to the denuded corneal surfaces (one eye of each patient) without sutures. RESULTS: Complete reepithelialization of the corneal surfaces occurred within one week in all four treated eyes. Corneal transparency was restored and postoperative visual acuity improved remarkably in all four eyes. During a mean follow-up period of 14 months, all corneal surfaces remained transparent. There were no complications. CONCLUSIONS: Sutureless transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells may be used to reconstruct corneal surfaces and can restore vision in patients with bilateral severe disorders of the ocular surface.

Aged↗

Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control.

We investigated physicochemical properties of two types of poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture polystyrene (TCPS) surfaces, to elucidate the influential factors for thermally regulated cell adhesion and detachment to PIPAAm-grafted surfaces. The two types of PIPAAm-grafted surfaces were prepared by the electron beam polymerization method. Attenuated total reflection Fourier transform infrared spectroscopy revealed that amounts of the grafted polymers were 1.4 +/- 0.1 microg/cm2 for PIPAAm-1.4 and 2.9 +/- 0.1 microg/cm2 for PIPAAm-2.9. Both PIPAAm-grafted surfaces showed hydrophobic/hydrophilic property alterations in response to temperature. However, PIPAAm-1.4 surfaces were more hydrophobic (cos theta = 0.21 at 37 degrees C and cos theta = 0.35 at 20 degrees C) than PIPAAm-2.9 (cos theta = 0.42 at 37 degrees C and cos theta = 0.50 at 20 degrees C) both above and below the PIPAAm's transition temperature. Thicknesses of the grafted PIPAAm layers were estimated to be 15.5 +/- 7.2 nm for PIPAAm-1.4 and 29.5 +/- 8.4 nm for PIPAAm-2.9, by the use of UV excimer laser and atomic force microscope. Bovine carotid artery endothelial cells (ECs) adhere to the surfaces of PIPAAm-1.4 and proliferate to form confluent cell monolayers. The cell monolayers were harvested as single cell sheets by temperature decrease from 37 to 20 degrees C. On the contrary, ECs did not adhere to the surfaces of PIPAAm-2.9. This phenomenon was correlated with an adsorption of cell adhesion protein, fibronectin, onto surfaces ofPIPAAm-1.4 and -2.9. In the case of nano-ordered thin grafted surfaces, the surface chain mobility is strongly influenced by the thickness of PIPAAm grafted layers because dehydration of PIPAAm chains should be enhanced by the hydrophobic TCPS surfaces. PIPAAm graft amounts, that is, thickness of the PIPAAm grafted layers, play a crucial role in temperature-induced hydrophilic/hydrophobic property alterations and cell adhesion/detachment behavior.

Acrylamides↗

Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.

A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.

Animals↗

Micropatterned surfaces prepared using a liquid crystal projector-modified photopolymerization device and microfluidics.

A commercial liquid crystal device projector was modified for photopolymerization using its on-board intense light source and a precision optical control circuit. This device projects reduced images generated by a typical personal computer onto the stage where photopolymerization on a surface occurs. This all-in-one device does not require expensive photomasks and external light sources. However, light scattering and diffraction through glass substrates resulted in undesired reactions in areas corresponding to masked (black) domains in mask patterns, limiting pattern resolution. To overcome this shortcoming, two-step surface patterning was developed. First, three-dimensional microstructures of crosslinked silicone elastomer were fabricated with this device and adhered onto silanized glass substrate surfaces, forming microchannels in patterns on the glass support. Then, acrylamide monomer solution containing photoreactive initiator was flowed into these micromold channels and reacted in situ. The resultant polyacrylamide layer was highly hydrophilic and repelled protein adsorption. Cell seeding on these patterns in serum-supplemented culture medium produced cells selectively adhered to different patterns: cells attached and spread only on unpolymerized silanized glass surfaces, not on the photopolymerized acrylamide surfaces. This technique should prove useful for inexpensive, rapid prototyping of surface micropatterns from polymer materials.

Acrylic Resins↗

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2.

Many types of organ-specific stem cells have been recently shown to exhibit a side population (SP) phenotype based on their ability to efflux Hoechst 33342 dye. Because stem cells from corneal epithelium reside in the basal layer of the limbal epithelium, the purpose of this study was to examine whether the limbal epithelium contains SP cells. The ATP-binding cassette transporter Bcrp1/ABCG2 is reported to contribute to the SP phenotype in cells from several diverse sources. Here we show data from fluorescence-activated cell sorting and real-time quantitative RT-PCR analysis showing that harvested limbal epithelial cells contain SP cells expressing ABCG2. Immunofluorescence revealed that a portion of limbal epithelial basal cells expressed ABCG2. Data indicate that ABCG2 positive limbal epithelial cells are putative corneal epithelial stem cells.

Adenosine Triphosphate↗

Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces.

The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from culture dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37 degrees C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to culture surfaces.

Animals↗

Temperature- and pH-responsive aminopropyl-silica ion-exchange columns grafted with copolymers of N-isopropylacrylamide.

We have designed copolymers of N-isopropylacrylamide, environmentally-responsive polymers, which respond to temperature and other external stimuli. In this study, we designed and synthesized copolymers that introduced ion-exchange groups. These copolymers responded to the temperature and the pH, and the copolymer-grafted aminopropyl silica beads were used as HPLC packing materials. This stationary phase altered the properties from hydrophilic to hydrophobic and from charge to non-charge by temperature and pH changes. We studied the separations of organic acids and phenylthiohydantoin-amino acids using environmentally-responsive chromatography, and confirmed the effects of the ion-exchange groups. The elution behaviors of these samples were controlled by the temperature changes without organic solvents in the mobile phase. It was confirmed that the interactions between the solute and stationary phase could be freely controlled by the temperature and the pH. Environmentally-responsive chromatography is expected to be applicable to the separation of pharmaceuticals and biomolecules, such as peptides, proteins and nucleic acids.

Acrylamides↗