VI MagnetobiologyCell interaction with nickel nanowiresToxicity studyAc field effect on on pre-osteoblastsIn vitro cell stimulation with strong pulse fieldsPlanned work

MANSE Midterm Review

Staff, PublicationsAdriele Prina-Mello Senior postdoc 2006-Fiona Byrne Postgrad 2006-Darragh Crotty Postgrad 2005-Nigel Carrol Experimental Officer

Collaborations: Yuri Volkov (TCD, Clinical Medicine)Ken Donaldson, Craig Poland (U. Edinburgh)

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Publications:

Internalization of ferromagnetic nanowires by different living cells, A. Prina-Mello, Zhu Diao and J. M. D. Coey, Journal of Nanobiotechnology 4:9 1-11 (2006) High content analysis of the biocompatibility of nickel nanowires. Fiona Byrne, A. Prina-Mello, A. Whelan, B.M. Mohamed, A. davies, Y. Gunko, J.M.D. Coey, Yuri Volkov, J. of Magnetism and Magnetic Material, (in press) Magnetic-fluorescent nanocomposites for biomedical multitasking S A. Corr, A. OByrne, Y. K. Gunko, S. Ghosh, D. F. Brougham, S. Mitchell, Y Volkov and A Prina-Mello, Chemical Communications, 4474 - 6 (2006) The evolution of chemotaxis assays from static models to physiologically relevant platforms S. Toetsch, P. Olwell, A. Prina-Mello and Y. Volkov, Integrative Biology in press (2009) In vivo EPR for dosimetry, H.M. Swartz, G. Burke and M. Coey, Radiation Measurements 42 SI 1075-1084 (2007)

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MANSE Proposal MagnetobiologyInvestigate the influence of static uniform magnetic fields and field gradients on cellular growth (proliferation, differentiation and death) and morphology (alignment and migration). For instance, study the influence of static magnetic fields on cellular growth and morphology, especially in relation to magnetohydrodynamics of trans-membrane ion flows

Manipulate and stimulate cells in novel ways, using some of the techniques and capabilities developed in CINSE, and in the electrochemistry section of the new program, specifically, nano and microscale magnet arrays and structured nanowires. For instance, develop magnetic methods of cellular manipulation and stimulation using custom-designed nanowires

Confluent MC3T3-E1 cells cultured with nickel nanowires, after magnetic separation and subsequent culture for 5 days.

Adhering cell with nanowireMANSE Magnetobiology proposal concept: Living Cells + Magnetic materials + External Field = Manipulation, Stimulation and Sensing Cell integration with magnetic nanowires

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Integration between living cells with magnetic carriers

Objective: develop standardised methodology for magnetic separation, alignment and manipulation on different cell lineages5 - 50 m

Cell-NW Interaction: Experimental Setup NW (diam. = 200 10 nm, length = 20 3 m)Cell used: MC3T3-E1 osteoblasts, UMR-106 rat osteoblasts and Marrow Stromal Cells (MSCs)Cell-NW binding/internalization: promoted by cellsCell-NW incubation time: overnightDevice used: 2 external Nd2Fe14B magnets ( >0.3 T)Cell separation time: 5 minutesAverage Cell-NW separation velocity: 400 m/sMagnetic torque applied to each cell during the alignment process mB 1.610-14 Nm. Cell adhesion force, calculated for human bone marrow stromal cell (HBMSC) (EHBMSC = 3 kPa, cell diam. 130 m) [2] Simon et al., (2003)[1] Prina-Mello et al, J. Nanobiotechnology (2006)

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Cell-NW interactionCell-NW separation and manipulation(Video)SEM of adherent MC3T3-E1 osteoblasts showing internalized nickel nanowireMSCs with internalized nickel nanowires

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Angular distributions of MSC+NW morphology in 0 field (left), and after alignment in a 100 mT applied field for 18 h (right)OrientationAngular distributions of Ni NW orientation in MSCs in zero field (left). After alignment in a 100 mT applied field for 18 h (right). 5 daysConfluent UMR-106 tumor cells

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Toxicity study[3] Byrne, Prina-Mello et al., (2009)[4] Prina-Mello et al., (2009)[5] Tian, Prina-Mello et al, (2008)[6] Whelan, Byrne, Prina-Mello et al., (2009)

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Need to quantify cytotoxicity due to:Nickel magnetic materials for in vitro/ in vivo use in biologyNanowire size, shape and concentrationPreparation method, batch to batch variability

Few studies have addressed these points in an unbiased, reproducible, and quantitative experimental studyObjective: To complete a full High Content Screening to address the i) time and ii) concentration toxic response dependence of Ni NWs High Content Screening (HCS) cell time-dependent response

HCS in a NutshellHCS cytotoxicity analysis (Bioinformatics protocol and algorithm):Computational load: N = 4 experimentsx 4 exposure time (3,6,24,72 hr) 3 Triplicates x each experiment x 9 field /well = Approx 9500 field measurements per each parameter[3] Byrne, Prina-Mello et al., (2009)

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[4] Prina-Mello et al., (2009) (in submission)Cell proliferation response to i) time and ii) concentration response

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Data courtesy of Poland and Donaldson

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AC field effect on pre-osteoblastsBackground

Literature suggests that EMF can affect osteoblast (bone cells) proliferation, differentiation and morphology [EMF 1,2,3,4,5,6]

Pulsed EMFs used clinically to enhance bone healing [EMF 6]

Objective Help understand if there is a scientific basis behind the fears concerning the carcinogenicity of 50/60 Hz AC electromagnetic fields as suggested by some previous epidemiological evidence

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ExperimentsPre-osteoblasts were exposed to homogenous 50 Hz, 2 mT AC EMF via solenoid in incubator at 37C for 24 hrs

Cells then fixed and fluorescently stained for F-Actin (cytoskeleton) and DNA (nuclei)

High content automated microscope analysis (Incell and Cellomics) was used to analyse image data

Cell morphology, proliferation, cell cycle and F-actin properties were examined

Changes in these are associated with bone formation and cancer development onsetMeasured outcomes: Student Award at the 30th Annual Meeting of The Bioelectromagnetics Society meeting in San Diego, CA.Selected as one of the Best images of 2007 GE Healthcare In Cell competition Showed in Times Square, NY

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High Content Screening ResultsThe field has no effect on the pre-osteoblast properties analysedMorphologyProliferationFActin Cytoskeleton Cell Cycle*[] Peer-reviewed manuscript in preparation

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In vitro cell stimulation with pulsed fieldsTranscranial Magnetic Stimulation (TMS)

Important experimental tool in Neuroscience used in clinically for treatment and diagnosis of neurological disorders such as Multiple Sclerosis and Parkinsons disease

How it works

Strong (1-2 T) magnetic pulse produced by TMS coilCircular electric field induced near coil centrePotential difference triggers the nerves or neurons firing via changes in their membrane electrical potential

ObjectivesTo extend the first demonstration of in vitro magnetic stimulation of neurons reported recently by Rotem et al. (2008)

To understand the physical and biochemical mechanisms involved in vitro

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ExperimentsProbability of firing dependent on geometry of neuronsAlignment with direction of induced EF => Probability of firingAlignment with Circular EF means neurons need to grow along rings

Controlling pattern of neuronal growth 1D culturePrevents neuronal adhesion on a ring-shaped channels of untreated surface to promoteneurons growth along the rings

Magnetic Stimulation:

Detection and analysis of neuronal firingNeurons stained with dye (Fluo-4) => Ca2+ binding markerNeuronal firing => Ca2+ influx => fluorescence intensityImaged via fast acquisition ccd cameraAnalysis software outputs intensity ([Ca2+]i) data for each neuron

Preliminary resultsDemonstrated electric stimulation (505 1700 V/m) of neuroblastoma cells (SH-SY5Y) using Ca2+ fluorescence imaging and Measured cell membrane depolarisation induced by applied EFNext step: Reliably imaging and measure magnetic stimulation on Neuroblastoma cellsElectrode stimulation of cells

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Conclusions, future plansThis work is developing valuable interdisciplinary relations within TCD and beyondMany attempts to detect magnetic field effects on cells are inconclusive or negativeNanowires have potential for cell labelling and manipulationComplete length/aspect ratio influence study on toxicityIn-vitro pulsed-field stimulation is a new area, which willbe developed

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