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VI Magnetobiology  Cell interaction with nickel nanowires  Toxicity study  Ac field effect on on pre-osteoblasts  In vitro cell stimulation with strong.

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Presentation on theme: "VI Magnetobiology  Cell interaction with nickel nanowires  Toxicity study  Ac field effect on on pre-osteoblasts  In vitro cell stimulation with strong."— Presentation transcript:

1 VI Magnetobiology  Cell interaction with nickel nanowires  Toxicity study  Ac field effect on on pre-osteoblasts  In vitro cell stimulation with strong pulse fields  Planned work

2 MANSE Midterm Review Staff, Publications Adriele Prina-Mello Senior postdoc Fiona Byrne Postgrad Darragh Crotty Postgrad Nigel Carrol Experimental Officer Collaborations: Yuri Volkov (TCD, Clinical Medicine) Ken Donaldson, Craig Poland (U. Edinburgh)

3 MANSE Midterm Review Publications: — Internalization of ferromagnetic nanowires by different living cells, A. Prina- Mello, Zhu Diao and J. M. D. Coey, Journal of Nanobiotechnology 4: (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. O’Byrne, Y. K. Gun’ko, S. Ghosh, D. F. Brougham, S. Mitchell, Y Volkov and A Prina-Mello, Chemical Communications, (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 (2007)

4 MANSE Midterm Review MANSE Proposal – Magnetobiology  Investigate 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

5 Confluent MC3T3-E1 cells cultured with nickel nanowires, after magnetic separation and subsequent culture for 5 days. Adhering cell with nanowire MANSE Magnetobiology proposal concept: Living Cells + Magnetic materials + External Field = Manipulation, Stimulation and Sensing Cell integration with magnetic nanowires

6 MANSE Midterm Review Integration between living cells with magnetic carriers Objective: develop standardised methodology for magnetic separation, alignment and manipulation on different cell lineages  m Ni nanowires

7 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 cells Cell-NW incubation time: overnight Device used: 2 external Nd 2 Fe 14 B magnets ( >0.3 T) Cell separation time: 5 minutes Average Cell-NW separation velocity: 400  m/s Magnetic torque applied to each cell during the alignment process mB≈ 1.6 ⋅ Nm. Cell adhesion force, calculated for human bone marrow stromal cell (HBMSC) (EHBMSC = 3 kPa, cell diam. ≅ 130 μm) [2] Simon et al., (2003) [1] Prina-Mello et al, J. Nanobiotechnology (2006)

8 MANSE Midterm Review Cell-NW interaction Cell-NW separation and manipulation (Video) SEM of adherent MC3T3-E1 osteoblasts showing internalized nickel nanowire MSCs with internalized nickel nanowires

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

10 MANSE Midterm Review Toxicity study NWs of different nature Shape Size Charge Stabilizers Functionality Phagocytes Epithelium Endothelium Fibroblasts ECM components Diagnostics Imaging Drug delivery Toxicology [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)

11 MANSE Midterm Review Need to quantify cytotoxicity due to: 1.Nickel magnetic materials for in vitro/ in vivo use in biology 2.Nanowire size, shape and concentration 3.Preparation method, batch to batch variability Few studies have addressed these points in an unbiased, reproducible, and quantitative experimental study Objective: 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

12 HCS in a Nutshell Automatic Data Archival Instantaneous Data DisplayAnalysis of Results Automated Plate DeliveryAuto-focus, Expose & AcquireAutomated Image Analysis Reader for 3 fluorescent dye staining: 1: Cell count (DAPI) 2: Cell permeability (FITC) 3: Lysosomal mass (TRITC) Automated multi-parameter analysis HCS cytotoxicity analysis (Bioinformatics protocol and algorithm): Computational load: N = 4 experiments x 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)

13 MANSE Midterm Review Nickel NW dose [ng/mL] Exposure time [hr] Cell proliferation reduction [%] CTRL Pos NiCl CTRL Pos NiSO [4] Prina-Mello et al., (2009) (in submission) Cell proliferation response to i) time and ii) concentration response

14 MANSE Midterm Review

15 Data courtesy of Poland and Donaldson

16 MANSE Midterm Review AC field effect on pre-osteoblasts Background 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

17 MANSE Midterm Review Experiments Pre-osteoblasts were exposed to homogenous 50 Hz, 2 mT AC EMF via solenoid in incubator at 37°C 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 onset Measured 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

18 MANSE Midterm Review High Content Screening Results The field has no effect on the pre-osteoblast properties analysed Morphology Proliferation F–Actin Cytoskeleton Cell Cycle *[] Peer-reviewed manuscript in preparation

19 MANSE Midterm Review In vitro cell stimulation with pulsed fields Transcranial Magnetic Stimulation (TMS) Important experimental tool in Neuroscience used in clinically for treatment and diagnosis of neurological disorders such as Multiple Sclerosis and Parkinson’s disease How it works Strong (1-2 T) magnetic pulse produced by TMS coil Circular electric field induced near coil centre Potential difference triggers the nerves or neurons firing via changes in their membrane electrical potential Objectives To 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

20 MANSE Midterm Review Experiments Probability of firing dependent on geometry of neurons Alignment with direction of induced EF => ↑ Probability of firing Alignment with Circular EF means neurons need to grow along rings Controlling pattern of neuronal growth 1D culture Prevents neuronal adhesion on a ring-shaped channels of untreated surface to promote neurons growth along the rings Magnetic Stimulation: Detection and analysis of neuronal firing Neurons stained with dye (Fluo-4) => Ca 2+ binding marker Neuronal firing => Ca 2+ influx => ↑ fluorescence intensity Imaged via fast acquisition ccd camera Analysis software outputs intensity ([Ca2+] i ) data for each neuron

21 Preliminary results Demonstrated electric stimulation (505 – 1700 V/m) of neuroblastoma cells (SH-SY5Y) using Ca 2+ fluorescence imaging and Measured cell membrane depolarisation induced by applied EF Next step: Reliably imaging and measure magnetic stimulation on Neuroblastoma cells Electrode stimulation of cells

22 MANSE Midterm Review Conclusions, future plans  This work is developing valuable interdisciplinary relations within TCD and beyond  Many attempts to detect magnetic field effects on cells are inconclusive or negative  Nanowires have potential for cell labelling and manipulation  Complete length/aspect ratio influence study on toxicity  In-vitro pulsed-field stimulation is a new area, which will  be developed

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