title: Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.
abstract: Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability.
authors.affiliation: Institute of Nanostructure Technologies and Analytics (INA), Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Voss
authors.affiliation: Department of Animal Physiology, Center of Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
authors.forename: HongYing
authors.initials: H
authors.lastname: Wei
authors.affiliation: Institute of Nanostructure Technologies and Analytics (INA), Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium.
authors.forename: Stuart
authors.initials: S
authors.lastname: Turner
authors.affiliation: Institute for Health and Consumer Protection, European Commission Joint Research Centre, Via E. Fermi 2749 TP125, I-21027 Ispra, VA, Italy.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Ceccone
authors.affiliation: Institute of Nanostructure Technologies and Analytics (INA), Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
authors.forename: Johann Peter
authors.initials: JP
authors.lastname: Reithmaier
authors.affiliation: Department of Animal Physiology, Center of Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Stengl
authors.affiliation: Institute of Nanostructure Technologies and Analytics (INA), Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany. Electronic address: popov@ina.uni-kassel.de.
authors.forename: Cyril
authors.initials: C
authors.lastname: Popov
mesh_terms: D000818:Animals; D001683:Biological Clocks; D002448:Cell Adhesion; D002478:Cells, Cultured; D002940:Circadian Rhythm; D003058:Cockroaches; D008297:Male; D008567:Membranes, Artificial; D058612:Nanodiamonds / Q000737:chemistry*; D009474:Neurons / Q000145:classification / Q000378:metabolism*
chemical_list: D008567:Membranes, Artificial; D058612:Nanodiamonds
keywords: Calcium imaging; Cell adhesion; Circadian neurons; Surface modification; Ultrananocrystalline diamond films