NUI Galway – UL Alliance First Annual ENGINEERING AND - ARAN ...

Development of a Micropipette Aspiration and Microscopy System to
Investigate Active Cytoskeletal Remodelling
Reynolds, N. 1 , McGarry J.P. 1
1 Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway
n.reynolds2@nuigalway.ie
Abstract
Remodelling of the active cytoskeleton plays a critical
role in the response of cells to mechanical stimuli.
Furthermore, the mechanical environment plays an
important role in cell differentiation [1]. Previous
studies have investigated the response of cells to
micropipette aspiration [2-4]. However, the active
response of the actin cytoskeleton has not been
considered. This study will investigate changes in the
actin cytoskeleton during micropipette aspiration of
spread and round cells. The experimental results will be
used to guide the development of an active formulation
of cytoskeletal remodelling in response to external
loading [5]. Detailed examination of the actin
cytoskeleton will also provide insight into remodelling
mechanisms.
1. Introduction
In order to elucidate the key biochemical processes
underlying the experimentally observed phenomena, it
is necessary to characterise dynamic changes in the
cytoskeleton. Micropipette aspiration in tandem with a
novel imaging technique will examine the evolving cell
microstructure under mechanical stimuli.
2. Materials and Methods
Computational: A finite element parametric study of
micropipette aspiration of viscoelastic cells was
performed. The effect of micropipette diameter, nucleus
diameter and vacuum pressure on cell aspiration length
was investigated.
Experimental: Cells will be aspirated with
micropipettes of 5, 10 and 20 µm internal diameter
attached to a custom built pressure control system. The
vacuum pressure applied to the micropipette is
generated from the pressure head difference between
points 1 and 2 on Fig 1.A. The pressure applied will be
controlled by adjusting the height Δh with a micromanipulator.
A damping chamber is used to link this
pressure to the micropipette so that fluctuations in
pressure are minimized.
A protected silver mirror (Thorlabs, Ltd.,
Cambridgeshire, UK) will be aligned at 45° to provide
an optical path that enables visualisations of
micropipette aspiration (Figure 1.B). A long range
objective lens accounts for the total distance between
the microscope (Olympus, IX-71 inverted microscope)
and the cell.
Osteoblast cells (MC3T3-E1) transfected with the
pGFP2-actin vector will be used to visualise the
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changing actin cytoskeleton in real time during the
experiment. Cells will be seeded onto glass slides for
0.5 and 3.0 hrs to examine the role of the cytoskeleton
in round and spread cell geometries respectively.
Figure 1: Schematic diagram of experiment setup (A)
including enlarged representation outlining the optical
path (B) and micropipette aspiration (C).
3. Results and Discussion
Computational results indicate that a micropipette
diameter greater than the nucleus diameter is desirable,
as well as vacuum pressure greater than 200 Pa.
Upon initial calibration of the pressure control
system, micropipette aspiration of spread and round
adhered cells will be completed. The experimental
results will be compared to a recent computational
model that includes active remodelling of the
cytoskeleton [5] Detailed examination of the actin
cytoskeleton will provide insight into cellular
mechanotransduction.
4. References
[1]. Discher et al, Science 310:1139-1143, 2005.
[2]. Chien et al, Biophysical Journal 24:436-487, 1987.
[3]. Trickey et al, J Orthop Res 22:131-139, 2004.
[4]. Thoumine et al, Eur Biophys J 28:222-234, 1999.
[5]. Ronan et al, ASME 2010 SBC, Naples, Fl, 2010.
Acknowlegments
Science Foundation Ireland-Research Frontiers Program
10/RFP/ENM2960