Download Bio-ultrasonics Group

Dr. Despina Bazou
Cardiff School of Biosciences
Connective Tissue Biology Laboratories
Cardiff
CF10 3US
UK
Tel: +44 (0)29 20 87 5158
Fax: +44 (0)29 20 87 4594
Research
Behaviour of cells in ultrasonic standing wave systems
Physical approaches such as optical trapping, dielectrophoresis, magnetic
labelling and ultrasound trapping offer means of manipulating cells in suspension.
Ultrasound trapping approaches are studies in our lab with a view to develop new
methodologies that can significantly improve the study of biological systems. The
technique employs a physical ultrasound standing wave trap (Fig. 1) that drives
particles, cells or droplets rapidly (less that 1 s) into a plane (the pressure nodal plane)
that is already in optical microscopic focus (Fig. 2). They then move within that
focussed plane to form 2- or 3-D aggregates that can be held and levitated in
suspension, for hours (Fig. 3).
Cell-cell interactions in suspension
The principal interest of our laboratory lies in understanding the cellular and
molecular mechanisms of cell-cell interactions with a specific focus on the temporal
progression of these interactions from the early stages of receptor engagement to
cytoskeletal organisation in a range of cell systems (neural progenitor, HepG2,
prostate epithelial and cancer cell lines, articular cartilage and chick mesenchymal
primary cells), using an ultrasound standing wave trap capable of levitating cells in
suspension, free of the substratum effects that are known to influence cell properties.
The function of the ultrasound trap is to hold cells close together so that the
likelihood of cell receptor interaction is increased very significantly over the
‘encounter in suspension’ situation. Significant numbers of interactions can take place
as an aggregate with a diameter of 1 mm contains approximately 5,000 cells. The
structure of the aggregate, the outcome of cell-cell interactions in a suspended
aggregate and their consequences for cell behaviour have been shown not to be
compromised by or dependent on the physical environment of the ultrasound trap.
We have also shown that the synchronous adhering cells, following membrane
receptor interactions, undergo the intracellular F-actin, catenin and connexin responses
on rapid time scales. Cells aggregated, spread their cell-cell contact interface and
developed confluent-culture-like F-actin patterns over the period of 1-30 min in the
trap (Fig. 4), as well as Cx43 distribution (Fig. 4) that was consistent with measured
gap junction functionality over the period of 1-60 min in the trap.
The potential of the ultrasound trap in the area of cancer biology has been also
recognised. We have shown that the ultrasound trap is a technique sensitive enough to
aid in the identification of the adhesive properties of cancer cells. Cancer cells
expressed proteins of the cadherin/catenin complex to a lesser extent than their noncancerous counterparts and as a result they were classified as less adhesive.
An integrated view of the development of a cell monolayer in the ultrasound
trap can be seen as a non biological concentration of cells under millimetre range
acoustic forces, a non-intrusive effect of the micron scale acoustic particle interaction
force that is small compared to the van der Waals interaction, acoustic-independent
cell receptor interactions that modify the short range interactions leading to adhesion
and then acoustic-independent progression of intracellular processes to form the
adhesively strong and communicating monolayer.
We believe that by unraveling the early molecular events involved in cell-cell
adhesion as well as the underlying molecular regulatory networks we will ultimately
understand
fundamental
biological
processes
such
as
tissue
construction,
differentiation, cancer development and metastasis.
A technique for the encapsulation of high cell density aggregates
The alginate encapsulation of cells to confine biomass within microcapsules
for cell grafts and its recent combination with biosil capsule coating is an active area
of research. We are interested in our group to develop a gel encapsulation technique
that departs from the minimum surface area to volume restriction of spherical
microcapsules and allows gelation of preformed cell aggregates reducing the need for
pre-graft incubation. The process involves forming a discoid cell aggregate in an
ultrasound standing wave resonator and then introducing an alginate/CaCl2 pre-gel into
the ultrasound trap where it preferentially sets about the cell aggregate. The discrete
encapsulated cell aggregates (discrete capsules) are discoid in shape and have a
thickness that allows accessibility to nutrient and gas exchange. The technique has
potential applications in the field of tissue engineering and the development of new
scaffold-free approaches used to repair, for example, cartilage defects as well as in the
development of in vitro biosensor device based on mammalian cells for real-time
toxicity analyses.
Recent publications
Bazou D, Coakley WT, Hayes AJ, Jackson SK. (2007) Long-term viability and
proliferation of alginate-encapsulated 3-D HepG2 aggregates formed in an ultrasound trap.
Submitted
Bazou D, Blain EJ, Coakley WT. (2007) NCAM and PSA-NCAM dependent membrane
spreading and F-actin reorganization in suspended adhering neural cells. In press
Kuznetsova L, Bazou D and Coakley WT. (2007) Stability of 2-D particle aggregates
held against flow stress in an ultrasound trap. In press
Edwards GO, Bazou D, Kuznetsova L, Coakley WT. (2007) Cell adhesion dynamics
and actin reorganisation in HepG2 cell aggregates. In press
Kuznetsova LA, and Coakley WT. (2006) Applications of ultrasound streaming and
radiation force in biosensors. In press
Bazou D, Dowthwaite GP, Khan IM, Archer CW, Ralphs JR, Coakley WT. (2006)
Gap junctional intercellular communication and cytoskeletal organization in
chondrocytes in suspension in an ultrasound trap. Mol Membr Biol. 2006 Mar-Apr;
23(2):195-205.
Khanna S, Hudson B, Pepper CJ, Amso NN, Coakley WT. (2006) Fluorescein
isothiocynate-dextran uptake by chinese hamster ovary cells in a 1.5 MHz ultrasonic
standing
wave
in
the
presence
of
contrast
agent.
Ultrasound Med Biol. Feb; 32(2):289-95.
Coakley WT, Bazou D. (2005) Particle and cell manipulation by radiation force in
ultrasound standing waves. In: Bubble and Particle Dynamics in Acoustic Fields:
Modern Trends and Applications (Ed. Alexander A. Doinikov). Research Signpost,
Transworld Research Network. Pp.313-338
Borthwick KA, Love TE, McDonnell MB, Coakley WT.(2005) Improvement of
immunodetection
of
bacterial
spore
antigen
by
ultrasonic
cavitation.
Anal Chem. Nov 15; 77(22):7242-5.
Kuznetsova LA, Martin SP, Coakley WT. (2005) Sub-micron particle behaviour and
capture at an immuno-sensor surface in an ultrasonic standing wave. Biosens
Bioelectron. Dec 15; 21(6):940-8.
Martin SP, Townsend RJ, Kuznetsova LA, Borthwick KA, Hill M, McDonnell MB,
Coakley WT. (2005) Spore and micro-particle capture on an immunosensor surface in
an ultrasound standing wave system. Biosens Bioelectron. Nov 15; 21(5):758-67.
Zourob M, Hawkes JJ, Coakley WT, Treves Brown BJ, Fielden PR, McDonnell MB,
Goddard NJ. (2005) Optical leaky waveguide sensor for detection of bacteria with
ultrasound attractor force. Anal Chem. Oct 1; 77(19):6163-8.
Bazou D, Foster GA, Ralphs JR, Coakley WT.(2005) Molecular adhesion
development in a neural cell monolayer forming in an ultrasound trap. Mol Membr
Biol. Jul-Aug; 22(4):373.
Bazou D, Kuznetsova LA, Coakley WT. (2005) Physical enviroment of 2-D animal
cell aggregates formed in a short pathlength ultrasound standing wave trap.
Ultrasound Med Biol. Mar; 31(3):423-30.
Gherardini L, Cousins CM, Hawkes JJ, Spengler J, Radel S, Lawler H, Devcic-Kuhar
B, Groschl M, Coakley WT, McLoughlin AJ. (2005) A new immobilisation method to
arrange particles in a gel matrix by ultrasound standing waves. Ultrasound Med Biol.
Feb; 31(2):261-72.
Kuznetsova LA, Khanna S, Amso NN, Coakley WT, Doinikov AA. (2005) Cavitation
bubble-driven cell and particle behavior in an ultrasound standing wave. J Acoust Soc
Am. Jan; 117(1):104-12.
Borthwick KA, Coakley WT, McDonnell MB, Nowotny H, Benes E, Groschl M.
Development of a novel compact sonicator for cell disruption. (2005)J Microbiol
Methods.Feb; 60(2):207-16.
Hawkes JJ, Barber RW, Emerson DR, Coakley WT. (2004) Continuous cell washing
and mixing driven by an ultrasound standing wave within a microfluidic channel.
Lab Chip. Oct; 4(5):446-52.
Bazou D, Coakley WT, Meek KM, Yang M, Pham DT (2004) Characterisation of the
morphology of 2-D particle aggregates in different electrolyte concentrations in an
ultrasound trap. Colloids Surf A. Phys Engineer Asp. Aug 20; 243(1-3): 97-104.
Coakley WT, Bazou D, Morgan J, Foster GA, Archer CW, Powell K, Borthwick KA,
Twomey C, Bishop J. (2004) Cell-cell contact and membrane spreading in an
ultrasound trap. Colloids Surf B Biointerfaces. Apr 15; 34(4):221-30.
Hawkes JJ, Long MJ, Coakley WT, McDonnell MB. (2004)Ultrasonic deposition of
cells on a surface. Biosens Bioelectron. Apr 15; 19(9):1021-8.
Morgan J, Spengler JF, Kuznetsova L, Coakley WT, Xu J, Purcell WM. (2004)
Manipulation of in vitro toxicant sensors in an ultrasonic standing wave. Toxicol In
Vitro. Feb; 18(1):115-20.
Khanna S, Amso NN, Paynter SJ, Coakley WT. (2003) Contrast agent bubble and
erythrocyte behavior in a 1.5-MHz standing ultrasound wave. Ultrasound Med Biol.
Oct; 29(10):1463-70.
Grant support
BBSRC
(with Prof. Archer and Dr. Ralphs)
‘Progression of cell interactions and intracellular processes triggered on contact in
an ultrasound trap’
DTI
(with AstraZeneca, HiMedica, QinetiQ, Uniscan Instruments, UWE, National Physics
Laboratory, Applied Enzyme Technology Ltd)
‘Development an in vitro biosensor device based on mammalian cells for real-time
toxicity analyses’
Collaborators
Internal
Professor Charlie W. Archer (CITER)
Professor Victor Duance (CITER)
Dr. James R. Ralphs (CITER)
Dr. Emma Blain (CITER)
Dr. George A. Foster (Neuroscience)
Professor Vincenzo Crunelli (Neuroscience)
Dr. Nazar Amso and Dr. Sanjay Khanna (Department of Obstetrics and gynaecology,
Cardiff Medical School, Heath Hospital)
Dr. Wen G. Jiang (Department of Surgery, Cardiff Medical School, Heath Hospital)
External
Professor Jean-Paul Thiery (ICMB-Singapore)
Dr. Yeh-Shiu Chu (Marie Curie Institute-Paris)
Figure 1: Schematic diagram of the cylindrical steel trap assembly, epi-microscope,
sample loading and ultrasound generation. Its main components were a 1.5 MHz disc
transducer attached to a steel acoustic coupling layer, a sample volume and a glass
acoustic reflector.
Figure 2: Schematic diagram of the temporal progression (from time zero (i) to less
than one second (ii) to tens of seconds (iii)) of aggregation of suspended cells in a
single pressure node half-wavelength ultrasound trap.
a)
b)
Figure 3: a) Neural cells suspended in the ultrasound trap 30 min after initiation of
ultrasound; a 2-D aggregate is formed (scale bar is 70 μm). b) HepG2 cells suspended
in the ultrasound trap 20 min after initiation of ultrasound; a 3-D aggregate is formed
(scale bar is 400 μm); pressure, 0.06 MPa.
1 min
60 min
Cx43
F-actin
Merged
Figure 4: Distribution of peripheral (a) and interfacial (d) Cx43, and short (b) and
long (e) interfacial F-actin in aggregates isolated from the trap after 1 (a, b) and 60 (d,
e) min of ultrasound exposure respectively: (c, f) superimposed images.