Poly D-lysine Research Articles

Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in Geobacter sulfurreducens

Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-d-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane.

A silk platform that enables electrophysiology and targeted drug delivery in brain astroglial cells

Astroglial cell survival and ion channel activity are relevant molecular targets for the mechanistic study of neural cell interactions with biomaterials and/or electronic interfaces. Astrogliosis is the most typical reaction to in vivo brain implants and needs to be avoided by developing biomaterials that preserve astroglial cell physiological function. This cellular phenomenon is characterized by a proliferative state and altered expression of astroglial potassium (K +) channels. Silk is a natural polymer with potential for new biomedical applications due to its ability to support in vitro growth and differentiation of many cell types. We report on silk interactions with cultured neocortical astroglial cells. Astrocytes survival is similar when plated on silk-coated glass and on poly- d-lysine (PDL), a well known polyionic substrate used to promote astroglial cell adhesion to glass surfaces. Comparative analyses of whole-cell patch-clamp experiments reveal that silk- and PDL-coated cells display depolarized resting membrane potentials (˜−40 mV), very high input resistance, and low specific conductance, with values similar to those of undifferentiated glial cells. Analysis of K + channel conductance reveals that silk-astrocytes express large outwardly delayed rectifying K + current (K DR). The magnitude of K DR in PDL- and silk-coated astrocytes is similar, indicating that silk does not alter the resting K + current. We also demonstrate that guanosine- (GUO) embedded silk enables the direct modulation of astroglial K + conductance in vitro. Astrocytes plated on GUO-embedded silk are more hyperpolarized and express inward rectifying K + conductance (K ir). The K + inward current increases and this is paralleled by upregulation and membrane polarization of K ir4.1 protein signal. Collectively these results indicate that silk is a suitable biomaterial platform for the in vitro studies of astroglial ion channel responses and related physiology.

Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts

CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.

Extracellular matrix produced by bone marrow stromal cells and by their derivative, SB623 cells, supports neural cell growth

Several studies have shown the benefits of transplanting bone marrow-derived multipotent mesenchymal stromal cells (MSC) into neurodegenerative lesions of the central nervous system, despite a low engraftment rate and the poor persistence of grafts. It is known that the extracellular matrix (ECM) modulates neuritogenesis and glial growth, but little is known about effects of MSC-derived ECM on neural cells. In this study, we demonstrate in vitro that the ECM produced by MSC can support neural cell attachment and growth. We also compare the neurosupportive properties of MSC to the MSC derivative, SB623 cells, which is being developed as a cell therapy for stroke. Embryonic rat brain cortical cells cultured for 3 weeks on human MSC- and SB623 cell-derived ECM exhibit about a 1.5 and 3 times higher metabolic activity, respectively, compared with the cultures grown on poly-D-lysine (PDL), although the initial neural cell adhesion to cell-derived ECM and PDL is similar. The MSC- and SB623 cell-derived ECM protects neural cells from nutrient and growth factor deprivation. Under the conditions used, only neurons grow on PDL. In contrast, both MSC- and SB623 cell-derived ECMs support the growth of neurons, astrocytes, and oligodendrocytes, as demonstrated by immunostaining. Morphologically, neurons on cell-derived ECM form more complex and extended neurite networks than those cultured on PDL. Together, these data indicate that the beneficial effect of MSC and SB623 cells in neurotransplantation could be explained in part by the neurosupportive properties of the ECM produced by these cells.

Growth Cone 3-D Morphology is Modified by Distinct Micropatterned Adhesion Substrates

The development, connectivity, and structural plasticity of neuronal networks largely depend on the directional growth of axonal growth cones (GCs). The morphology and 3-D profile of axons and GCs of primary hippocampal neurons, grown onto glass surfaces coated with poly-D-lysine (PDL) and micropatterned with stripes of the adhesion molecule L1 by using the indirect microcontact printing, were investigated. Neurons were fixed at early stages (one to seven days) of in vitro development prior to synapse formation, and analyzed by fluorescence and atomic force microscopy. The latter technique allowed us to investigate the 3-D morphology of the GCs, and detect their morphological rearrangements during axon outgrowth and during contact with the underlying substrate. We found that axons decreased their height-to-width ratio over development in culture, and that this value became particularly low when the axon and the GC proceeded onto a surface containing attracting cues such as L1 with respect to GCs growing onto a nonspecific adhesion substrate such as PDL. Along with this shape change of the axons, GCs lying onto L1 tracks displayed a flattened shape, ideal for sensing and progression, whereas GCs onto areas of nonspecific adhesion displayed more prominent shapes and steeper edges.

Positioning and guidance of neurons on gold surfaces by directed assembly of proteins using Atomic Force Microscopy

We demonstrate that Atomic Force Microscopy nanolithography can be used to control effectively the adhesion, growth and interconnectivity of cortical neurons on Au surfaces. We demonstrate immobilization of neurons at well-defined locations on Au surfaces using two different types of patterned proteins: 1) poly- d-lysine (PDL), a positively charged polypeptide used extensively in tissue culture and 2) laminin, a component of the extracellular matrix. Our results show that both PDL and laminin patterns can be used to confine neuronal cells and to control their growth and interconnectivity on Au surfaces, a significant step towards the engineering of artificial neuronal assemblies with well-controlled neuron position and connections.

Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex

In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly- d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3β (GSK-3β) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.

Nerve Cell Patterning Using Polytetrafluoroethylene and Poly-D-Lysine

We developed a patterning technique for nerve cells that uses polytetrafluoroethylene (PTFE) and poly-D-lysine (PDL). The PDL layer on the PTFE substrate acted as a buffer for nerve cell adhesion, while Ar ion-irradiated areas on PTFE were used for separation of nerve cells. This structure was then used as a scaffold. A matrix circuit of nerve cells with dendrites or axons was subsequently observed on the scaffold after a week of cell culture. The Ar ion-irradiated area exhibited graphite or amorphous carbon, and PDL was detached from carbon grains by mechanical stress during cell culture. A possible explanation for the adhesion between PDL and PTFE is the van der Waals force. The stress between PDL/PTFE was reduced by the flexibility of PDL gel, which prevented separation of PDL from PTFE.

Application Forum: Improving Cell-Based Biotechnological Screening Approaches Using the Novel Advanced TC™ Cell Culture Surface

BioTechniquesVol. 45, No. 5 Application Forum - Sponsored PaperOpen AccessImproving Cell-based Biotechnological Screening Approaches Using the Novel Advanced TC™ Cell Culture SurfaceLara MarchettiLara MarchettiGreiner Bio-One GmbH, Frickenhausen, GermanySearch for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000113018AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionBiotechnology is often used to refer to the genetic engineering technology of the 21st century. Combining disciplines like genetics, molecular biology, biochemistry, and cell biology, biotechnology can also be defined as an all-encompassing life science.Focussing on cellular screening approaches using immortalized cell lines, primary cells or co-culture models, cell based assays are currently one of the most important biotechnological techniques. Researchers in both the academic and industrial markets are increasingly adopting a cell-based approach, shifting their focus away from biochemical analysis of discrete cellular components to the complex analysis of an in vivo like system. Therefore the utilization of living cells leads to more comprehensive data, which complement results from earlier biochemical assays.Obstacles of In vitro Cell CultureIn vivo cells of a multi-cellular organism are embedded in the three-dimensional structure of the extracellular matrix (ECM) of adjacent cells. In addition to providing structural support, the ECM also comprises a wide range of cellular growth factors and mediates biochemical signals which essentially influence cellular proliferation and survival.Contrariwise, cultivation of cells in vitro mainly refers to a two-dimensional culture on plastic surfaces lacking the vital signals provided by the connective tissue.Optimizing Cellular AdhesionOne approach to optimize cellular adhesion is to coat matrix-specific proteins to the plastic surfaces simulating an ECM-like surrounding to the cell. Poly-D-Lysine (PDL), Poly-L-Lysine (PLL), fibronectin, laminin and collagen are the commonly used proteins for these types of coatings.An entirely new development is the imitation of the cellular surrounding in a non-biological way. These innovative surfaces feature many advantages.Due to the production process the modification of the polymer assures a constant, reproducible product quality. Additionally, transport and storage can be carried out at room temperature. Due to the non-biological origin, the surface is not endangered by degradation and stable for several years. Likewise, the possibility of cross reaction or contamination based on the biological protein can be circumvented.The novel Advanced TC™ polymer modification positively influences cellular features and functions. Enhanced cell attachment and higher proliferation rates improve cell expansion (Figure 1), facilitating cultivation of sensitive and primary cells as well as cells cultivated under restricted growth conditions.Figure 1. Accelerated proliferation of SKNMC cells.SKNMC cells were seeded in a 96-well plate with a concentration of 20,000 cells/well, cultivated at 37°C and 5% CO2 for the indicated time period and stained with crystal violet to visualize adherent cells. Due to the improved adherence and accelerated proliferation increased cell numbers can be detected on the Advanced TC™ surface.Furthermore, the Advanced TC™ surface facilitates consistent and homogenous cell attachment, increasing the overall cell yield and reducing cell loss during processes such as the automated washing steps of high-throughput applications.A positive effect on cell morphology is particularly apparent during cultivation of sensitive cells, serum deprivation, or after cellular stress induced by transfection or transduction processes. Moreover, cells cultivated on the Advanced TC™ surface exhibit higher transgene activity after the described gene transfer.ConclusionThe novel Advanced TC™ cell culture surface improves cell adherence, leading to consistent and homogenous cell attachment, an in vivo–like morphology, and minimized cellular detachment during media changes or washing steps. It facilitates the cultivation of fastidious and sensitive cells as well as usage of serum-reduced or serum-free media. The optimal cultivation conditions accelerate proliferation, increase cell yield and maximize transgene activity in transfected cells. In summary, the innovative Advanced TC™ technology optimizes assay consistency of cell-based biotechnological screening approaches.FiguresReferencesRelatedDetails Vol. 45, No. 5 Follow us on social media for the latest updates Metrics History Published online 16 May 2018 Published in print November 2008 Information© 2008 Future Science LtdPDF download

The glia-derived extracellular matrix glycoprotein tenascin-C promotes embryonic and postnatal retina axon outgrowth via the alternatively spliced fibronectin type III domain TNfnD

Tenascin-C (Tnc) is an astrocytic multifunctional extracellular matrix (ECM) glycoprotein that potentially promotes or inhibits neurite outgrowth. To investigate its possible functions for retinal development, explants from embryonic day 18 (E18) rat retinas were cultivated on culture substrates composed of poly-d-lysine (PDL), or PDL additionally coated with Tnc or laminin (LN)-1, which significantly increased fiber length. When combined with LN, Tnc induced axon fasciculation that reduced the apparent number of outgrowing fibers. In order to circumscribe the stimulatory region, Tnc-derived fibronectin type III (TNfn) domains fused to the human Ig-Fc-fragment TNfnD6-Fc, TNfnBD-Fc, TNFnA1A2-Fc and TNfnA1D-Fc were studied. The fusion proteins TNfnBD-Fc and to a lesser degree TNfnA1D-Fc were stimulatory when compared with the Ig-Fc-fragment protein without insert. In contrast, the combination TNfnA1A2-Fc reduced fiber outgrowth beneath the values obtained for the Ig-Fc domain, indicating potential inhibitory properties. The monoclonal J1/tn2 antibody (clone 578) that is specific for domain TNfnD blocked the stimulatory properties of the TNfn-Fc fusions. When postnatal day 7 retinal ganglion cells were used rather that explants, Tnc and Tnc-derived proteins proved permissive for neurite outgrowth. The present study highlights a strong retinal axon growth-promoting activity of the Tnc domain TNfnD, which is modulated by neighboring domains.

Laminin activates NF-κB in Schwann cells to enhance neurite outgrowth

Extracellular matrix (ECM) molecules and Schwann cells (SCs) are important components of peripheral nerve regeneration. In this study, the role of the transcription factor nuclear factor kappa B (NF-kappaB) in SC activation in response to laminin and the subsequent effect on in vitro neurite outgrowth was investigated. Immunocytochemistry and Western blot analysis showed that compared with poly-d-lysine (PDL), laminin enhanced the phosphorylation of IkappaB and p65 NF-kappaB signalling proteins in SCs. Phospho NF-kappaB-p65 was localised to the nucleus indicating activation of NF-kappaB. To assess the functional effect of NF-kappaB activation, SCs plated on PDL or laminin were pre-treated with NF-kappaB inhibitors, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) or Z-leu-leu-leu-CHO (MG-132) before NG108-15 neuronal cells were seeded on the SC monolayer. After 24h co-culture in the absence of inhibitors, SCs seeded on laminin enhanced the mean number and length of neurites extended by NG108-15 cells (1.87+/-0.13 neurites; 238.74+/-8.53microm) compared with those cultured in the presence of SCs and PDL (1.26+/-0.07 neurites; 157.57+/-9.80microm). At 72h, neurite length had further increased to 321.83+/-6.60microm in the presence of SCs and laminin. Inhibition of NF-kappaB completely abolished the effect of laminin on SC evoked neurite outgrowth at 24h and reduced the enhancement of neurite length by over 60% at 72h. SC proliferation was unaffected by NF-kappaB inhibition suggesting that the NF-kappaB signalling pathway plays a discrete role in the activation of SCs and their neurotrophic potential.

Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells

BackgroundInteractions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.ResultsA reproducible protocol was used to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESCs. This defined adherent culture system allowed us to examine the effect of ECM molecules on neural differentiation of hESCs. hESC-derived differentiating embryoid bodies were plated on Poly-D-Lysine (PDL), PDL/fibronectin, PDL/laminin, type I collagen and Matrigel, and cultured in neural differentiation medium. We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees. Glia did not appear until 4 weeks later. Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a dose-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by the antibody against integrin α6 or β1 subunit.ConclusionWe defined laminin as a key ECM molecule to enhance neural progenitor generation, expansion and differentiation into neurons from hESCs. The cell-laminin interactions involve α6β1 integrin receptors implicating a possible role of laminin/α6β1 integrin signaling in directed neural differentiation of hESCs. Since laminin acts in concert with other ECM molecules in vivo, evaluating cellular responses to the composition of the ECM is essential to clarify further the role of cell-matrix interactions in neural derivation of hESCs.

Automated image analysis of neuroblastoma cell networks cultured on microelectrode array plates

Event Abstract Back to Event Automated image analysis of neuroblastoma cell networks cultured on microelectrode array plates Heidi Teppola1*, Jyrki Selinummi1, Jertta-Riina Sarkanen1, Antti Pettinen1, Tuula O. Jalonen2, Olli Yli-Harja1 and Marja-Leena Linne1 1 Tampere University of Technology, Finland 2 University of Jyväskylä, Finland In order to form functional, long-term neuronal networks of cells for electrophysiological microelectrode array recordings (MEA; MultiChannel Systems, Reutlingen, Germany), the first important step is to optimize cell growth on MEA plates. In this work, human SH-SY5Y neuroblastoma cells were cultured on MEA plates treated with coating agents poly-D-lysine (PDL), poly-L-lysine (PLL), polyethyleneimine (PEI) with laminin, and laminin alone. Additionally, to differentiate the SH-SY5Y cells to form electrically active long-term networks, they were also treated with all-trans-retinoic acid (RA) and cholesterol (CHOL). The SH-SY5Y cell line was selected as test application because of its differentiation to neuron-like phenotype and its active synaptic vesicle recycling after RA+CHOL treatment. The ultimate goal of this work was to find the best alternative of the various coating agents by following cell growth, changes in morphology, and the level of differentiation. Instead of manual inspection cell growth was estimated by specifically developed image processing algorithms which automatically detect the area occupied by cells from each of the microscopy images. First, the image was segmented into small regions using Quad-tree segmentation (Jain R. et al. 1995, Machine Vision, McGraw-Hill, Inc., NY). These regions were then, based on the intensities and size of the regions, labeled into four classes: electrodes, cells, background, and artifacts (see figure). The analysis procedure was implemented in the eCognition 5.0 image analysis platform (Definiens AG, München, Germany). The effects of the coating treatments on cell differentiation and the presence of synaptic communication on MEA plates were verified with AM1-43 fluorescent staining of the synaptic vesicle recycling. The results show that neuroblastoma cells attach on MEA plates with all of the used coating agents, but based on the automated analysis there is less cell growth with PEI+laminin coating and with no coating. Furthermore, the maturation, morphology, and distribution of the cells are more neuron-like with PEI+laminin than with any other treatments. We conclude that the MEA coating agents have a strong impact on cell morphology, growth, and viability. Based on our results, more emphasis should be put on the role of coating agents in determining the structure, function, and electrophysiological properties of the created neuronal networks. For computational modeling purposes there is an increasing need for means to clarify the relationships between the structure and the synaptic connectivity of a neural network, as well as the complexity of recorded microelectrode array signals. The here presented automated image analysis procedure supports the design of structurally relevant biological neural networks for modeling. Beecher Instruments Inc, Sun Prairie, WI, USA is acknowledged for guidance in image analysis. tn_image 3 conference image 3 conference Conference: Neuroinformatics 2008, Stockholm, Sweden, 7 Sep - 9 Sep, 2008. Presentation Type: Poster Presentation Topic: Neuroimaging Citation: Teppola H, Selinummi J, Sarkanen J, Pettinen A, Jalonen TO, Yli-Harja O and Linne M (2008). Automated image analysis of neuroblastoma cell networks cultured on microelectrode array plates. Front. Neuroinform. Conference Abstract: Neuroinformatics 2008. doi: 10.3389/conf.neuro.11.2008.01.063 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Jul 2008; Published Online: 25 Jul 2008. * Correspondence: Heidi Teppola, Tampere University of Technology, Tampere, Finland, heidi.teppola@tuni.fi Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Heidi Teppola Jyrki Selinummi Jertta-Riina Sarkanen Antti Pettinen Tuula O Jalonen Olli Yli-Harja Marja-Leena Linne Google Heidi Teppola Jyrki Selinummi Jertta-Riina Sarkanen Antti Pettinen Tuula O Jalonen Olli Yli-Harja Marja-Leena Linne Google Scholar Heidi Teppola Jyrki Selinummi Jertta-Riina Sarkanen Antti Pettinen Tuula O Jalonen Olli Yli-Harja Marja-Leena Linne PubMed Heidi Teppola Jyrki Selinummi Jertta-Riina Sarkanen Antti Pettinen Tuula O Jalonen Olli Yli-Harja Marja-Leena Linne Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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In vitro growth and differentiation of canine olfactory bulb-derived neural progenitor cells under variable culture conditions

The dog serves as a large animal model for multiple neurologic diseases that may potentially benefit from neural progenitor cell (NPC) transplantation. In the adult brain, multipotent NPCs reside in the subventricular zone and its rostral and caudal extensions into the olfactory bulb and hippocampus. The olfactory bulb represents a surgically accessible site for obtaining cells for autologous NPC transplantation. To model conditions that would occur for ex vivo gene therapy in the postnatal brain, NPCs were isolated from the canine olfactory bulb, expanded ex vivo under different culture conditions, and compared quantitatively for growth and immunophenotype. Under standard growth conditions, canine olfactory bulb-derived NPCs (OB-cNPCs) could be expanded nearly 500-fold in the time evaluated. Canine OB-cNPCs grown on poly- d-lysine (PDL) or on PDL-fibronectin had similar growth rates, whereas supplementation with leukemia inhibitory factor (LIF) resulted in significantly slower growth. However, when OB-cNPC cultures were grown on PDL-fibronectin or PDL supplemented with LIF, a greater proportion of cells with neuronal markers were generated upon differentiation.

Characterization of surface modification on microelectrode arrays for in vitro cell culture

This study aims to investigate surface-modified microelectrodes on the microelectrode arrays (MEAs) for neuronal interfaces with in vitro cell culture. The polyimide (PI) MEA was fabricated by using micro-electro-mechanical systems (MEMS) techniques. Self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) were utilized to modify the microelectrode surface of the MEA. The SAMs' modified surface of microelectrodes offered a reliable interface to immobilize biological ligands through covalent bonding. To increase biocompatibility, the poly-D-lysine (PDL) was immobilized on the SAMs' modified microelectrodes. Several analytical techniques were used to define the physical structure and functional groups of surface-modified gold microelectrodes on the MEA. Spectra of the Fourier transform infrared reflection (FTIR) were applied to characterize the molecular structure of MUA-SAMs and PDL on the microelectrodes. The spectra, two peaks of amide I (at 1,613 cm(-1)) and amide II (at 1,548 cm(-1)), revealed that covalent amide bonding existed in PDL-MUA-SAMs modified surfaces. The thickness and formation of the MUA and PDL were also observed and quantified by using an atomic force microscope (AFM). The impedance measurement of PDL-MUA-SAMs modified MEA only increased slightly to an average of 524.6 +/- 55.8 kOmega from 352.9 +/- 34.4 kOmega of bare gold microelectrode (p < 0.05, N = 20). In addition, the time-course changes of total impedance resulting from cell sealing resistance and gap reactance were recorded for 7 days for inferring the growth of cell lines on the electrode contact of modified MEA. The experiment of 3T3 fibroblasts, PC12 cells, primary glial cells, and primary cortical neurons cultured on the modified MEAs displayed a good adhesion rate. These biocompatibility assays demonstrated that the neuronal cells are able to grow in a proximity to PDL-MUA-SAMs modified microelectrodes of the MEAs for effective electrophysiological stimulation/sensing schemes and for future implantation purposes.

Mitofusin 2 Protects Cerebellar Granule Neurons against Injury-induced Cell Death

Of the GTPases involved in the regulation of the fusion machinery, mitofusin 2 (Mfn2) plays an important role in the nervous system as point mutations of this isoform are associated with Charcot Marie Tooth neuropathy. Here, we investigate whether Mfn2 plays a role in the regulation of neuronal injury. We first examine mitochondrial dynamics following different modes of injury in cerebellar granule neurons. We demonstrate that neurons exposed to DNA damage or oxidative stress exhibit extensive mitochondrial fission, an early event preceding neuronal loss. The extent of mitochondrial fragmentation and remodeling is variable and depends on the mode and the severity of the death stimuli. Interestingly, whereas mitofusin 2 loss of function significantly induces cell death in the absence of any cell death stimuli, expression of mitofusin 2 prevents cell death following DNA damage, oxidative stress, and K+ deprivation induced apoptosis. More importantly, whereas wild-type Mfn2 and the hydrolysis-deficient mutant of Mfn2 (Mfn2(RasG12V)) function equally to promote fusion and lengthening of mitochondria, the activated Mfn2(RasG12V) mutant shows a significant increase in the protection of neurons against cell death and release of proapoptotic factor cytochrome c. These findings highlight a signaling role for Mfn2 in the regulation of apoptosis that extends beyond its role in mitochondrial fusion.

Neuronal Network Morphology and Electrophysiologyof Hippocampal Neurons Cultured on Surface-Treated Multielectrode Arrays

Toward the development of biocompatible surfaces for implantable electrode arrays and the creation of patterned neuronal networks, the impact of select biochemical substrates [poly-D-lysine (PDL), polyornithine (PO), polyethylenimine (PEI), and a basement membrane extract (BM)] on network morphology and spontaneous electrophysiological activity of dissociated hippocampal neurons was investigated. Cultured in serum-free Neurobasal medium at 100 000 cells/cm(2), neurons attached to each substrate. PDL, PO, and PEI induced little or no neuronal clustering and process fasciculation, whereas the addition of BM promoted these features. The ratios of somas to processes, and axons to dendrites, as determined by immunohistochemical staining and image analysis were comparable across all substrates. Spontaneous firing was recorded using planar multielectrode arrays (MEAs) at the third week in vitro for the two most divergent morphologies according to Euclidian cluster analysis, namely those induced by PO + BM and PEI. Mean spike amplitude, mean firing rate, median interspike interval (ISI), mean burst rate, and correlation index were analyzed and compared to morphological features. Synchronized bursting was highly correlated with neuronal clustering and process fasciculation. Spike amplitude was negatively correlated with thin branching which was most evident in neurons grown on PEI. These data indicate that factors, which influence adherence of neurons to surfaces, can profoundly impact both neuronal network morphology and electrophysiological activity in vitro.

Macrophage behavior on multilayered DNA‐coatings in vitro

A pivotal factor to consider in the development of biomaterials and biomaterial coatings is the inflammatory response to these materials. The insertion of implants is followed by protein adsorption and subsequent interactions with cellular components of the biological surroundings, in which macrophages play a dominant role through the production of a myriad of signaling molecules. In view of this, the aims of the present study were to evaluate (i) gross protein adsorption to, and (ii) in vitro behavior of macrophages on novel biomaterial coatings, composed of poly-D-lysine (PDL) or poly(allylamine hydrochloride) (PAH) and DNA, and to compare these coatings with negative (noncoated glass) and positive controls (noncoated glass + LPS-stimulation). The results demonstrate that multilayered DNA-coatings do not affect gross protein adsorption compared to noncoated controls. Cell culture experiments showed that the attachment to, and viability and morphology of two types of macrophages cultured on multilayered DNA-coatings is comparable to noncoated controls. Still, macrophages repeatedly showed decreased secretion levels of the proinflammatory cytokine TNF-alpha on multilayered DNA-coatings, whereas no differences were observed in the secretion of IL-1beta, IL-10, and TGF-beta1. Appropriate animal studies are required to elucidate if these in vitro indications have clinical effects on the inflammatory and wound healing processes around implants.

スプレーパターニングと可動式微小電極による小規模神経回路の位置選択的自発活動計測

In order to precisely grasp the self-organization process of functional neuronal circuits from individual immature neurons, electrical activity of neuronal circuits should be continuously recorded with their well-defined inner structures and boundary conditions. Standing on this viewpoint, we have developed a series of practical experimental methods for the non-invasive recording of electrical activity from cultured small neuronal circuits by combining a simple micropatterning method of cultured neurons and a site-selective extracellular recording method. The micropatterning was demonstrated with a commercially available spray, and thousands of small neuronal circuits were formed in a 35-mm polystyrene dish by spraying PDL (poly-D-lysine) solution onto the BSA (bovine serum albumin)-coated culture surface. These small neuronal circuits consisted of several neurons and kept well-patterned for more than two weeks. The site-selective recording was demonstrated by means of a handmade mobile microelectrode, and spontaneous firings were detected at multiple recording sites in a small neuronal circuit. This series of experimental methods can be directly applicable to the investigation into the developmental process of the morphology and the functions of various electrically excitable multicellular organisms.

Differential Expression of the B′β Regulatory Subunit of Protein Phosphatase 2A Modulates Tyrosine Hydroxylase Phosphorylation and Catecholamine Synthesis

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.