3D Spheroids’ Sensitivity to Electric Field Pulses Depends
on Their Size
Received: 19 December 2012 / Accepted: 8 March 2013 / Published online: 22 March 2013
Ó Springer Science+Business Media New York 2013
Abstract Dramatic differences of cells behavior exist
between cells cultured under classical 2D monolayers and
3D models, the latter being closer to in vivo responses.
Thus, many 3D cell culture models have been developed.
Among them, multicellular tumor spheroid appears as a
nice and easy-to-handle 3D model based on cell adhesion
properties. It is composed of one or several cell types and is
widely used to address carcinogenesis, or drugs screening.
A few and recent publications report the use of spheroids to
investigate electropermeabilization process. We studied the
response of spheroids to electrical ﬁeld pulses (EP) in
terms of their age, diameter or formation technique. We
found that small human HCT-116 colorectal spheroids are
more sensitive to electric ﬁeld pulses than larger ones.
Indeed, the growth of spheroids with a diameter of 300 lm
decreased by a factor 2 over 4 days when submitted to EP
(8 pulses, lasting 100 ls at a 1,300 V/cm ﬁeld intensity).
Under those electrical conditions, 650 lm spheroids were
not affected. These data were the same whatever the for-
mation method (i.e. hanging drop and nonadherent tech-
niques). These observations point out the fact that
characteristics of 3D cell models have to be taken into
account to avoid biased conclusions of experimental data.
Keywords 3D Á Electrochemotherapy Á
Electropermeabilization Á Growth Á Size Á Spheroid
Accumulating evidences underline that 3D cell models are
superior to classical 2D cell culture to mimic and predict
in vivo situations (Ghajar and Bissell 2010; Nyga et al.
2011; Pampaloni et al. 2007). Indeed, some studies found
differences in drug sensitivity (Chitcholtan et al. 2012),
proliferation (dit Faute et al. 2002), extracellular matrix
production (Kelm et al. 2010) and gene expression proﬁle
(Ghosh et al. 2005) between 2D and 3D in vitro cell cultures.
In that context, Sutherland (1988) developed in the 1980s
a 3D cellular model devoid of any exogenous material named
spheroid. It is based on cell aggregation properties. Spheroid
formation can be initiated in a microgravity environment
within a bioreactor (Marrero et al. 2009), using spinner ﬂask
or gyratory shaker cultures (Canatella et al. 2004), with the
hanging drop technique (Kelm et al. 2003), or by seeding
cells in nonadherent 96-well plates (Wenger et al. 2005)orin
96-well plates coated with agarose (Ma et al. 2012) or poly-
HEMA (Ivascu and Kubbies 2006). In spheroid, cells
develop intercellular junctions and secrete extracellular
matrix components such as collagens, laminin, ﬁbronectin
and glycosaminoglycans (Kunz-Schughart et al. 2006;
Nederman et al. 1984; Stevens et al. 2009). Spheroid appli-
cations are widely developed to assess invasion (Hattermann
et al. 2011), angiogenesis (Correa de Sampaio et al. 2012),
chemotherapeutic agents screening (Vinci et al. 2012), novel
drug delivery systems (Kim et al.
2010), cell–cell interac-
tions (Santini et al. 2000), and to investigate therapeutic
potential of treatment (Upreti et al. 2011).
Among all the delivery methods, a physical approach
named electropermeabilization, or electroporation, is now
accepted in clinics (Kee et al. 2011; Matthiessen et al. 2012;
Mir et al. 1998; Sersa et al. 2011). This technique showing that
cell membrane can be efﬁciently transiently permeabilized
under application of electric pulses has been introduced by
Neumann in the 1970s (Neumann and Rosenheck 1972).
L. Gibot Á M.-P. Rols
IPBS-CNRS, 205 route de Narbonne, BP64182, F-31077
L. Gibot Á M.-P. Rols (&)
de Toulouse, UPS, 31077 Toulouse, France
J Membrane Biol (2013) 246:745–750