ARUP; ISBN: 978-0-9562121-5-3 - CMBBE 2012 - Cardiff University

lifestyle and obesity. In that context, we have recently found that 3T3-L1 adipocyte
cultures subjected to large substrate tensile strains produce intracytoplasmic lipids at a
faster rate and to a larger extent (Levy et al., 2011, Shoham et al., 2011b). In another
study, reduction of adipogenesis in murine stromal cells following cyclic strain was
found to be magnitude dependent: loading with 2% strain resulted in partial inhibition
compared to 10% strain, and loading with 0.5% strain could not inhibit adipogenesis at
all (Huang et al., 2011). Combining these, the magnitude of the mechanical strain
applied to the adipocytes is an important factor influencing the nature of the adipogenic
response. Considering also that the stiffness of the cells directly influence on the extent
of cellular deformations when adipose tissues are weight-bearing, it is important to
characterize stiffness distributions of differentiating adipocytes.
Stiffness of cells can be assessed using a variety of different methods, e.g. atomic force
microscopy (AFM), micropipette aspiration or optical tweezers (Callies et al., 2009;
Hochmuth, 2000). In the AFM, the most widely used technique, a tip is pressed against
the cell surface so that the membrane is indented. This deviates the AFM cantilever
which serves as a soft spring. Then a force versus deformation curve is calculated and
the slope of the curve reflects the stiffness (Callies et al., 2009). The weaknesses of this
technique include thermal fluctuation masking of forces that are of the order of 10-15
pN. Furthermore, the variable shape of a typical AFM probe determines the nature of
the force-deformation curve (Hochmuth, 2000). In the micropipette aspiration
technique, the surface of a cell is aspirated into a small tube while tracking the leading
edge of its surface. Interpretation of the measurements with basic continuum models
leads to values for a cell's elastic and viscous properties (Hochmuth, 2000). The
drawbacks of this method include a drift in the null (zero) setting for the pressure and
the demand for training and skill on the part of the experimenter (Hochmuth, 2000). The
optical tweezers method imposes a controlled force and deformation on micro object
using a laser light, but is generally limited to the measurement of small forces on the
order of 50 pN (Hochmuth, 2000). Additionally, all of these techniques are either
complex and require integrating particles in the experimental system or allow
measurement of the effective stiffness of the entire cell, whereas better understanding of
the adipocyte mechanics will be acquired by measuring the stiffness distribution within
the cell, in its deferent regions. In this study, we introduce a novel method of nondestructive
imaging and analysis of adipocyte cultures undergoing differentiation, for
evaluating parametric effective stiffness properties of the cells as well as of the intracellular
lipid droplets. Our results are relevant to research of adipose-related diseases,
particularly overweight and obesity.
3. METHODS
3.1 Cell culturing
Mouse embryonic 3T3-L1 cells (American Type Culture Collection, ATCC) were
cultured in a growth medium (GM) consisting of high-glucose Dulbecco's modified
eagle medium (DMEM, 4.5mg/ml; Biological Industries, Israel), 10% fetal bovine
serum (FBS, Biological Industries), 1% L-glutamine (Biological Industries), 0.1%
Penicillin-Streptomycin (Pen-Strep; Sigma, Israel) and 0.5% 4-(2-hydroxyethyl)-1piperazineethanesulfonic
acid (HEPES; Sigma). The maximum confluence allowed for
the cultures was ~80% prior to passaging. When a confluence of 90% was achieved,
2