Rodriguez-Losada, NoelaRomero, PabloEstivill-Torrús, GuillermoGuzmán de Villoria, RobertoAguirre, Jose A2017-05-262017-05-262017-03-23Rodriguez-Losada N, Romero P, Estivill-Torrús G, Guzmán de Villoria R, Aguirre JA. Cell survival and differentiation with nanocrystalline glass-like carbon using substantia nigra dopaminergic cells derived from transgenic mouse embryos. Plos One. 2017; 12(3):e0173978.http://hdl.handle.net/10668/2664Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Because the interest in carbon nanomaterials has grown within the last decade in light of a wide variety of applications, the aim of this study was to test and evaluate the suitability and cytocompatibility of a particular nanometer-thin nanocrystalline glass-like carbon film (NGLC) composed of curved graphene flakes joined by an amorphous carbon matrix. This material is a disordered structure with high transparency and electrical conductivity. For this purpose, we used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryos. Cells were cultured either in a powder of increasing concentrations of NGLC microflakes (82±37μm) in the medium or on top of nanometer-thin films bathed in the same culture medium. The metabolism activity of SN4741 cells in presence of NGLC was assessed using methylthiazolyldiphenyl-tetrazolium (MTT) and apoptosis/necrosis flow cytometry assay respectively. Growth and proliferation as well as senescence were demonstrated by western blot (WB) of proliferating cell nuclear antigen (PCNA), monoclonal phosphorylate Histone 3 (serine 10) (PH3) and SMP30 marker. Specific dopaminergic differentiation was confirmed by the WB analysis of tyrosine hydroxylase (TH). Cell maturation and neural capability were characterized using specific markers (SYP: synaptophysin and GIRK2: G-protein-regulated inward-rectifier potassium channel 2 protein) via immunofluorescence and coexistence measurements. The results demonstrated cell positive biocompatibility with different concentrations of NGLC. The cells underwent a process of adaptation of SN4741 cells to NGLC where their metabolism decreases. This process is related to a decrease of PH3 expression and significant increase SMP30 related to senescence processes. After 7 days, the cells increased the expression of TH and PCNA that is related to processes of DNA replication. On the other hand, cells cultured on top of the film showed axonal-like alignment, edge orientation, and network-like images after 7 days. Neuronal capability was demonstrated to a certain extent through the analysis of significant coexistence between SYP and GIRK2. Furthermore, we found a direct relationship between the thickness of the films and cell maturation. Although these findings share certain similarities to our previous findings with graphene oxide and its derivatives, this particular nanomaterial possesses the advantages of high conductivity and transparency. In conclusion, NGLC could represent a new platform for biomedical applications, such as for use in neural tissue engineering and biocompatible devices.enAnimalesApoptosisWestern blottingCarbonoLínea celularReplicación del ADNConductividad eléctricaCélulas madre embrionariasCitometría de flujoTécnica del anticuerpo fluorescenteGrafitoHistonasRatones transgénicosNanoestructurasCanales de potasioAntígeno nuclear de célula en proliferaciónMedicina regenerativaSerinaSustancia negraSinaptofisinaIngeniería de tejidosTirosina 3-MonooxigenasaMedical Subject Headings::Organisms::Eukaryota::AnimalsMedical Subject Headings::Phenomena and Processes::Cell Physiological Phenomena::Cell Physiological Processes::Cell Death::ApoptosisMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Electrophoresis::Blotting, WesternMedical Subject Headings::Chemicals and Drugs::Inorganic Chemicals::Elements::CarbonMedical Subject Headings::Anatomy::Cells::Cells, Cultured::Cell LineMedical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::DNA ReplicationMedical Subject Headings::Phenomena and Processes::Physical Phenomena::Magnetic Phenomena::Electromagnetic Phenomena::Electricity::Electric ConductivityMedical Subject Headings::Anatomy::Cells::Stem Cells::Embryonic Stem CellsMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Clinical Laboratory Techniques::Cytological Techniques::Cell Separation::Flow CytometryMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Clinical Laboratory Techniques::Cytological Techniques::Histocytochemistry::Immunohistochemistry::Fluorescent Antibody TechniqueMedical Subject Headings::Chemicals and Drugs::Inorganic Chemicals::Elements::Carbon::GraphiteMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Nuclear Proteins::HistonesMedical Subject Headings::Organisms::Eukaryota::Animals::Animal Population Groups::Animals, Genetically Modified::Mice, TransgenicMedical Subject Headings::Technology and Food and Beverages::Technology, Industry, and Agriculture::Manufactured Materials::NanostructuresMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Carrier Proteins::Membrane Transport Proteins::Ion Channels::Potassium ChannelsMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Nuclear Proteins::Proliferating Cell Nuclear AntigenMedical Subject Headings::Disciplines and Occupations::Health Occupations::Medicine::Regenerative MedicineMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Amino Acids::Amino Acids, Neutral::SerineMedical Subject Headings::Anatomy::Nervous System::Central Nervous System::Brain::Mesencephalon::Tegmentum Mesencephali::Substantia NigraMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Membrane Proteins::Vesicular Transport Proteins::SynaptophysinMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Culture Techniques::Cell Engineering::Tissue EngineeringMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Metalloproteins::Iron-Binding Proteins::Nonheme Iron Proteins::Tyrosine 3-Monooxygenaseresearch article28334019open access10.1371/journal.pone.01739781932-6203PMC5363826