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
Staff, Publications
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•
•
•
Adriele Prina-Mello Senior postdoc 2006Fiona Byrne Postgrad 2006Darragh Crotty Postgrad 2005Nigel Carrol Experimental Officer
• Collaborations: Yuri Volkov (TCD, Clinical Medicine)
• Ken Donaldson, Craig Poland (U. Edinburgh)
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 4:9 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. O’Byrne, Y. K. Gun’ko, 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)
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 transmembrane 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
MANSE Midterm Review
Cell integration with magnetic nanowires
Adhering cell
with nanowire
Confluent MC3T3-E1 cells
cultured with nickel nanowires,
after magnetic separation and
subsequent culture for 5 days.
MANSE Magnetobiology proposal concept:
Living Cells + Magnetic materials + External Field = Manipulation, Stimulation and
Sensing
Integration between living cells with magnetic carriers
5 - 50 m
Objective: develop standardised methodology for magnetic
separation, alignment and manipulation on different cell
lineages
Ni
nanowires
MANSE
Midterm Review
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 Nd2Fe14B 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⋅10-14 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)
Cell-NW interaction
MSCs with internalized nickel nanowires
Cell-NW separation and manipulation
(Video)
SEM of adherent MC3T3-E1 osteoblasts
MANSE Midterm Review
showing internalized nickel nanowire
Confluent UMR-106 tumor cells
Orientation
5 days
Angular distributions of Ni NW orientation in MSCs in zero
field (left). After alignment in a 100 mT applied field for 18 h
(right).
Angular distributions of MSC+NW morphology in 0 field (left),
and after alignment in a 100 mT applied field for 18 h (right)
MANSE Midterm Review
Toxicity study
NWs of different nature
Shape
Diagnostics
Size
Imaging
Charge
Drug delivery
Toxicology
Stabilizers
Functionality
Phagocytes
Epithelium
Endothelium
Fibroblasts
ECM components
MANSE Midterm Review
[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)
High Content Screening (HCS) cell time-dependent
response
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
MANSE Midterm Review
HCS in a Nutshell
Automated Plate Delivery
Auto-focus, Expose & Acquire
Automated Image Analysis
Reader for 3 fluorescent dye staining:
1: Cell count (DAPI)
2: Cell permeability (FITC)
of Results
Instantaneous
Dataprotocol
Displaymass
Automatic Data Archival
3: Lysosomal
(TRITC)
HCSAnalysis
cytotoxicity
analysis (Bioinformatics
and
algorithm):
Computational load:
N = 4 experiments
Automated multi-parameter analysis
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)
Cell proliferation response to i) time and ii) concentration response
MC3T3-E1 cell proliferation w & w/o Ni Nw vs Exposure time
1.4
y = -0.0403x3 + 0.2166x2 - 0.3386x + 1.1306
R² = 1
CTRL
Nickel NW
dose
[ng/mL]
Exposure time
[hr]
Cell proliferation
reduction
[%]
4.2
42
420
2100
CTRL Pos
NiCl2
CTRL Pos
NiSO4
72
72
72
72
72
34
43
59
84
73
72
62
1.2
Normalised cell count
4.2 ng/mL
1
42 ng/mL
0.8
0.6
y = -0.0615x3 + 0.3567x2 - 0.7013x + 1.4086
R² = 1
420 ng/mL
0.4
0.2
2100 ng/mL
y = 0.0487x3 - 0.358x2 + 0.515x + 0.7081
R² = 1
NiCl2
0
3hr
6hr
24hr
Exposure time [hr]
72hr
NiSO4
MANSE Midterm Review
[4] Prina-Mello et al., (2009) (in submission)
MANSE Midterm Review
Data courtesy of Poland and Donaldson
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
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.
Review
Selected as one of the Best images ofMANSE
2007 GEMidterm
Healthcare
In Cell competition – Showed in Times Square, NY
High Content Screening Results
Proliferation
Morphology
Cell Cycle
F–Actin Cytoskeleton
The field has no effect on the pre-osteoblast properties analysed
MANSE Midterm Review
*[] Peer-reviewed manuscript in preparation
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
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) => Ca2+ binding marker
Neuronal firing => Ca2+ influx => ↑ fluorescence intensity
Imaged via fast acquisition ccd camera
Analysis software outputs intensity ([Ca2+]i) data for each neuron
MANSE Midterm Review
Preliminary results
Demonstrated electric stimulation
(505 – 1700 V/m) of neuroblastoma
cells (SH-SY5Y) using Ca2+
fluorescence imaging and
Measured cell membrane
depolarisation induced by applied EF
Change in Fluorescence vs Time
12
10
Electrode stimulation of cells
Next step:
Reliably imaging and measure
magnetic stimulation on
Neuroblastoma cells
F - F0
8
6
4
2
0
-2
0
30
60
90
120
Time (secs)
150
180
210
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
MANSE Midterm Review