Article

Stiffness of MDCK II Cells Depends on Confluency

and Cell Size
Stefan Nehls,1 Helen Nöding,1 Susanne Karsch,1 Franziska Ries,1 and Andreas Janshoff1,*
1
€ t Göttingen, Institute of Physical Chemistry, Göttingen, Germany
Georg-August-Universita

ABSTRACT Mechanical phenotyping of adherent cells has become a serious tool in cell biology to understand how cells
respond to their environment and eventually to identify disease patterns such as the malignancy of cancer cells. In the steady
state, homeostasis is of pivotal importance, and cells strive to maintain their internal stresses even in challenging environments
and in response to external chemical and mechanical stimuli. However, a major problem exists in determining mechanical prop-
erties because many techniques, such as atomic force microscopy, that assess these properties of adherent cells locally can
only address a limited number of cells and provide elastic moduli that vary substantially from cell to cell. The origin of this spread
in stiffness values is largely unknown and might limit the significance of measurements. Possible reasons for the disparity are
variations in cell shape and size, as well as biological reasons such as the cell cycle or polarization state of the cell. Here, we
show that stiffness of adherent epithelial cells rises with increasing projected apical cell area in a nonlinear fashion. This size
stiffening not only occurs as a consequence of varying cell-seeding densities, it can also be observed within a small area of
a particular cell culture. Experiments with single adherent cells attached to defined areas via microcontact printing show that
size stiffening is limited to cells of a confluent monolayer. This leads to the conclusion that cells possibly regulate their size
distribution through cortical stress, which is enhanced in larger cells and reduced in smaller cells.

INTRODUCTION
Cellular biophysics with emphasis on the mechanical prop-
erties of adherent cells has received increasing attention
over the past decades, as multiple techniques have shown
that biomechanics is involved in a variety of processes
such as cell spreading (1,2), migration (3,4), proliferation,
and stem cell differentiation (5–7) and is altered depending
on the malignancy of certain types of cancer (8,9). One way
to assess the mechanical properties of cells is to externally
deform them with a defined force or pressure and measure
the response function. Viscoelastic responses to external
mechanical stimuli are found to originate mainly from the
actin cortex attached to the plasma membrane (10). Defor-
mation of cells either globally or locally with a sharp
indenter is usually interpreted either in terms of a contact
mechanics model, providing the Young’s modulus, or in
terms of a tension model, in which cortical tension prevails
at low strain, whereas area dilatation dominates at large
strain (11,12). Here, we focus on the apical mechanics of
adherent epithelial cells in terms of contact mechanics, in
which studies have already shown that the thin actomyosin
Submitted November 27, 2018, and accepted for publication April 22, 2019.
*Correspondence: ajansho@gwdg.de
Editor: Christopher Yip.
https://doi.org/10.1016/j.bpj.2019.04.028
Ó 2019 Biophysical Society.
cortex, together with the plasma membrane of such cells,
dominates the repulsive forces that are experienced by nano-
indenters (13,14). In epithelial cells, mechanical homeosta-
sis is of major importance and must continue even in
challenging environments, e.g., high or low osmolarity, to
ensure layer integrity (15). Disruption of this integrity by
the creation of defects in the cell layer leads to altered me-
chanical properties of the remaining cells surrounding the
defect. This change holds for multiple spheres of influence
up to several tens of micrometers away from the defect
(16–19).
Based on these findings, information on mechanical
changes within a layer may be naturally transmitted between
cells and poses an ideal way of establishing and maintaining
mechanical homeostasis within the layer. Homeostasis of
cellular morphology within monolayers is a prerequisite
for their functionality. Although epithelial cells like the
MDCK II cell line show high proliferation rates under sub-
confluent conditions, their growth is restricted upon reaching
confluency by contact inhibition (20,21). Although this
might seem like epithelial morphogenesis is finished in early
confluent monolayers of these cells, further development
persists even for days after reaching confluency, during
which cell-cell contacts are strengthened and supercellular
structures like domes or tubules are being formed (22,23).

2204 Biophysical Journal 116, 2204–2211, June 4, 2019


Although downregulated, cell division still takes place and
depends on the area occupied by the dividing cell, with larger
cells exhibiting higher division rates (24).
In this study, we show that there exists a strong correla-
tion between cellular stiffness and the cells’ projected apical
area, i.e., the occupied space within a cell monolayer. Our
finding points toward a regulation of cell size in a confluent
layer driven by cortical tension. Knowledge of this correla-
tion between cell stiffness and projected cell area permits us
to estimate and potentially also to eliminate the often-
observed variation in mechanical measurements due to a
variation in cell size. We suggest following a precise proto-
col to ensure that comparable data sets are gathered, in
which variations in seeding density and incubation time
are kept at a minimum.
MATERIALS AND METHODS
Cell culture
Madin Darby canine kidney cells (MDCK II; Health Protection Agency,
Salisbury, UK) were cultivated in minimal essential medium (MEM with
Earl’s salts, 2.2 g/L NaHCO3; Biochrom, Berlin, Germany) containing
4 mM L-glutamine (Lonza, Basel, Switzerland) and 10% (v/v) fetal calf
serum (FCS; BioWest, Nuaille, France). Stem and samples were kept at
37
C and 7.5% CO2 (Heracell 150i; Thermo Fisher Scientific, Waltham,
MA). Subcultivation was performed using standard culture flasks (TPP, Tra-
sadingen, Switzerland) by addition of 0.05% trypsin and 0.02% EDTA
(Biochrom) and short incubation to remove the adherent cells from the cul-
ture surface. Suspended cells were mixed with FCS and centrifuged, and the
pellet was dissolved in MEM and seeded into fresh flasks.
Cells destined to serve in experiments of confluent cells were taken in the
last step. 220 cells per square millimeter (c/mm2) were seeded into petri
dishes (Ibidi, Martinsried, Germany) and kept at 37
C and 7.5% CO2 for
48 h, unless stated otherwise. Before experiments, cell media were
exchanged to MEM containing HEPES (15 mM on confluent cells, 40 mM
on single cells, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Bio-
chrom) as well as 0.2 mg/mL penicillin/streptomycin (PAA, Pasching, Ger-
many) and 0.25 mg/mL amphotericin B (Biochrom).
Cell patterning
To create micrometer-scale patterns on culture dishes, plasma-induced
protein patterning was used. First, patterns were drawn in AutoCAD
(Autodesk, San Rafael, CA), and the corresponding mask was created
(Compugraphics, Jena, Germany). The passivated, microstructured wafers
were then used to cast polydimethylsiloxane (SYLGARD 184; Dow
Corning, Wiesbaden, Germany) molds by mixing polymer and curing agent
in a 10:1 ratio and curing it at 70
C for 4 h. The finished stamp was peeled
off the wafer.
Glass-bottomed petri dishes (Ibidi) were cleaned by washing with ultra-
pure water and ethanol and dried in nitrogen stream. The freshly cured pol-
ydimethylsiloxane stamp was cut into 5  5 mm pieces and placed onto the
glass surface. While in contact, the dish was placed in a plasma cleaner and
exposed to oxygen plasma for 90 s. Immediately after plasma treatment, a
solution of poly-L-lysine-graft-poly-ethyleneglycol (0.5 mL, PLL (20)-g
[3.5]-PEG (2)/ tetramethylrhodamine; SuSoS, D€ubendorf, Switzerland)
was added to each individual stamp, and the sample rested for 1 h at
23
C under exclusion of light. Within the given time period, the solvent
evaporated. After incubation, the stamps were removed, and the dish was
washed three times with phosphate-buffered saline (PBS; Biochrom)
Cell Size Mechanics
without Mg2þ or Ca2þ and a solution of bovine collagen I (0.2 mg/mL;
Thermo Fisher Scientific) in PBS was added and incubated for another
2 h. Finally, the dish was again washed three times with PBS and twice
with MEM, and 100,000 MDCK II cells suspended in 2.5 mL M10F40
(MEM containing 10% FCS (Biowest), 4 mM L-glutamine (Lonza), peni-
cillin/streptomycin (0.2 mg/mL; PAA, Pasching, Germany), 0.25 mg/mL
amphotericin B (Biochrom), and 40 mM HEPES (Biochrom)) were added
and incubated at 37
C. After 60 min, the sample was rinsed with M10F40 to
remove any nonadherent cells, and atomic force microscopy (AFM) mea-
surements were started.
AFM
Experiments were performed using MLCT cantilevers (knorm ¼ 0.01
pN/nm; Bruker AFM Probes, Camarillo, CA) on an atomic force micro-
scope (either MFP-3D Origin; Asylum Research, Santa Barbara, CA or
NanoWizard III; JPK, Berlin, Germany). Samples were kept at 37
C in
pH-buffered media during all experiments. Force maps with a resolution
of 1–8 mm2 per force-distance curve (FDC) were acquired. To determine
the Young’s moduli, cells were indented up to a force of 0.5–1 nN at vertical
speeds of 2–5 mm/s. To receive the size of individual cells and correlate that
to their Young’s modulus, slope maps were calculated from the force-
distance data by baseline correcting and linear fitting of the lowest 20 nm
of the data. In confluent cells, cell-cell contacts are much stiffer than central
cell regions, and high slopes therefore mark contact lines (see Figs. S1 and
S2). These boundaries of cell-cell contacts were then chosen manually and
used for cell segmentation. In single-cell studies, cell bodies were chosen in
a similar fashion based on the substrate showing extremely steep slopes in
the FDCs.
To determine viscoelastic properties, the cantilever, which has been in-
dented into the cellular cortex to a depth of about 1 mm, was actively oscil-
lated to examine the impact of the cytoskeleton (9,25,26). The cantilever
was exited to sinusoidal oscillations at different frequencies ranging from
5 to 100 Hz with small amplitudes. The complex shear modulus G*(u)
can then be obtained by the amplitude diminution and the phase shift be-
tween the in- and output signal. The real part of the resulting complex shear
modulus, the storage modulus G0 (u), describes the frequency-dependent
elastic properties, whereas the imaginary part, the loss modulus G00 (u), dis-
plays the energy dissipation in the system and represents its viscous prop-
erties. The fraction h(u) ¼ G00 (u)/G0 (u) is the so-called loss tangent, a
model free parameter describing the material properties of the cell (9).
The complex shear modulus G* increases with frequency following a
weak power law with an exponent b usually in between 0.2 and 0.4. The
loss modulus G00 exhibits lower values than G0 in the low-frequency regime
(<50 Hz). Hence, in this regime, MDCK II cells behave more elastically
rather than viscous. The mild power law and the elastic nature of the cell
justifies our approach using Young’s moduli from fitting a purely elastic
model to the data. In contrast, at larger frequencies, the cells display
more fluid-like properties. A successful attempt to explain this power law
behavior in G* in the sense of an active soft glassy material has been sug-
gested by Fabry and co-workers (27,28). The idea is based on the soft glassy
rheology model, assuming that the cytoskeleton of the cell is held together
by weak attractive forces between neighboring elements (28,29). Therefore,
the complex shear modulus G* of living cells is fitted with the power law
structural damping (Eq. 1) using a complex, nonlinear, least-squares fitting
routine:

p    b
bp u

G ¼ G0 Gð1  bÞcos b 1 þ i tan þ ium;
2 2 u0
(1)
with G0 the scaling factor describing the stiffness of the sample; b,
the power-law coefficient; u0, the scaling factor of the frequency (set to
1 rad/s); and the viscosity m. G0 can be easily converted in the apparent
Young’s modulus E0 by E0 ¼ 2G0(1 þ n), where n is Poisson’s ratio
Biophysical Journal 116, 2204–2211, June 4, 2019 2205
Nehls et al.

(assumed to be 0.3 because cells consist predominately of water, which is necting proteins such as ZO-1, F-actin, or E-cadherin were
incompressible). Notably, the scaling factor of the frequency is set arbi- visually inspected (see Fig. S4). The mechanical properties
trarily, which prevents a direct comparison between the Young’s modulus
obtained from force indentation data using a purely elastic contact
of the cells were obtained from microrheology experiments
mechanics model. as described previously (9,32).
We investigated layers originating from three different
seeding densities (270, 540, and 815 c/mm2). Each sample
Theory was incubated for 48 h, with a clear tendency of smaller
cell sizes for higher seeding densities. On these samples,
Cellular mechanics measured by external probes is often described in terms
of contact mechanics such as the Hertz model, considering the deformation
AFM-based microrheology was performed to probe the
of a semi-infinite fully elastic space by a rigid indenter. Such deformations elastic and viscous responses as a function of cell size.
are usually characterized by a single parameter describing the elastic Essentially, two parameters were obtained from the micro-
response, the so-called Young’s modulus E. Employing a conical indenter rheological spectra: the apparent Young’s modulus, E0,
with half-opening angle q results in the following approximate relationship which describes the elastic stiffness (Fig. 1, top), and the
between the force f and the indentation depth d (30):
loss tangent h, which provides information about the rela-
2E tanðqÞ 2 tion of the viscous and elastic properties at a given fre-
f ¼ d: (2)
pð1  n2 Þ quency (Fig. 1, bottom). We have chosen 100 Hz because
it turned out in a previous study that at this frequency, differ-
In typical force spectroscopy experiments, a force setpoint fSP is given,
and indentation is continued until the specified force setpoint is reached; ences between different cell lines are most pronounced (9).
hence, all acquired data share roughly the same maximal force but different The loss tangent is a quantitative measure of to what extent
indentation depths. The equation above is only true during contact between
probe and samples and does not apply to regions of the FDC without con-
tact. To evaluate FDCs without knowledge of the contact point, which is
usually difficult to determine automatically, we use the integral of the whole
curve, which is invariant to the length of the noncontact region.
Z dSP
2E tanðqÞ 2
Welast ¼ d dd (3)
0 pð1  n2 Þ
We know that indentation depth at the force setpoint equals
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
fSP pð1  n2 Þ
dSP ¼ : (4)
2E tanðqÞ
Together with Eq. 3, the Young’s modulus can be obtained from the
elastic energy Welast via
3
fSP  
E ¼ 2
p 1  n2 : (5)
18 Welast tanðqÞ

This idea of obtaining the elastic modulus automatically from FDCs

without determining the contact point is similar to the force integration to
equal limits, which has been applied to extract relative values (31). Howev-
er, here, we actually obtain the absolute Young’s modulus. The procedure is
strictly correct for ideal data, but the addition of noise might lead to a biased
modulus even for large sampling. For typical FDCs, at least, as they occur
in our experiments, we can show that from simulated and noise-decorated
FDCs, the correct modulus is found with high precision (see Fig. S3).

RESULTS
Cell mechanics as a function of seeding density
Our first approach was to seed different amounts of MDCK
II cells on equally sized petri dishes to obtain confluent cell
monolayers with different individual cell size. Seeding high
numbers of cells leads to an early and dense confluent layer
and consequently to a small average cell size. To verify that
a confluent monolayer was formed at each cell-seeding den-
sity, fluorescence stainings of important cell-cell intercon-
FIGURE 1 Young’s modulus E0 (top) and loss tangent at 100 Hz (bottom)
of MDCK II seeded at different cell densities. Box plots extend from the
25th to the 75th percentile, and whiskers from the 10th to the 90th. Individ-
ual data points are shown as dots; some outliers are not shown. Four
different cell-seeding densities were used, these being 190, 270, 540 and
815 c/mm2. Cells were allowed to grow for 48 h before measurement, and
in the case of 190 c/mm2, for 72 h. Number of viscoelastic spectra: n ¼
4092 (>20 cells), 7789 (>20 cells), 2027 (>20 cells), and 1622 (>12 cells),
respectively. A rank-sum test was performed to test the null hypothesis that
the data of the indicated data sets are from populations with equal medians.
Asterisks indicate that the null hypothesis was rejected at the 0.05% (***)
significance level. To see this figure in color, go online.

2206 Biophysical Journal 116, 2204–2211, June 4, 2019


the viscous properties (G00 ) prevail over the elastic properties
(G0 ). For a seeding density of 540 c/mm2, a median Young’s
modulus of E0(540 c/mm2) ¼ 850 Pa was found, which is in
good agreement with previously reported Young’s moduli of
the same cell line under similar culture conditions (E0(330–
540 c/mm2) ¼ 560 Pa (32)). For higher seeding densities,
the Young’s modulus decreased to E0(815 c/mm2) ¼ 460
Pa, whereas lower seeding densities resulted in a higher
elastic modulus of E0(270 c/mm2) ¼ 1.0 kPa. Hence, higher
seeding densities generate smaller cells correlating with
lower Young’s moduli. Notably, E0 obtained from rheolog-
ical data is not directly comparable with the Young’s
modulus from fitting a pure elastic contact model to the
data because E0 scales with the arbitrarily set time t0, here
set to 1 s.
The loss tangent computed at 100 Hz does not follow a
systematic trend. For all samples, the loss tangent was found
to be h > 1; thus, G00 > G0 . Comparing the lowest and highest
cell-seeding densities used in this study, we observed a slight
increase from h(270 c/mm2) ¼ 2.04 to h(815 c/mm2) ¼ 2.14.
However, intermediate seeding densities showed an overall
lower loss tangent of h(540 c/mm2) ¼ 1.86.
To further explore this relationship, we decided to
perform additional experiments with a seeding density of
only 190 c/mm2. Here, the cells grow so sparsely that an in-
cubation time of 3 days was necessary to obtain a confluent
monolayer. However, after incubation, we found even larger
cells compared to the other seeding conditions, and the
Young’s modulus was indeed increased in these samples
(Fig. 1). We also observed a significant drop of the loss
tangent to h(190 c/mm2) ¼ 1.68, which indicates a more
solid-like behavior of these larger cells. This is expected
because more prestressed cells are exhibiting a smaller po-
wer-law factor, which indicates a less fluid behavior. The
elastic modulus follows the general trend that larger cells
display a larger stiffness, i.e., E0(190 c/mm2) ¼ 1.3 kPa.
We rationalize this trend by considering that neighboring
cells pull more strongly at larger cells, forcing them into a
highly prestressed state.
These findings underline the importance of adhering
rigidly to established protocols with respect to both cell-
seeding density and growth time when extracting mechani-
cal properties from cell culture samples. In fact, we suggest
that evaluation of cell density at the time of experiments
should be performed and the data supplied in biomechanical
studies. The question was now whether the observed effect
was due to variations in cell size or, for instance, polarity of
the cells.
Size-dependent elasticity of confluent cells
The variation in mechanical behavior of cells in monolayers
could be caused by several aspects that might change during
layer maturation. Even after establishment of a confluent
monolayer, cell density still increases for days, continuously
Cell Size Mechanics
decreasing the mean area that cells occupy within the layer
(21,23). To test whether the difference in area on its own is
responsible for the variation in stiffness, we conducted ex-
periments in which we acquired force maps across multiple
cells within one particular sample and determined the area
of each individual cell. By doing so, we can circumvent
effects that are caused by different lengths of postconfluent
incubation periods. However, because this includes the
acquisition and evaluation of large data sets, we used a fully
automated analysis in the following, yielding solely the
Young’s modulus as the description of the cellular elastic
behavior. This is further justified by the finding that viscous
contribution did not change much with seeding density.
To determine the size distribution of cells under our
experimental conditions as precisely as possible, we stained
for the tight junction protein ZO-1 to facilitate cell segmen-
tation. Employing automated analysis of fluorescence
micrographs over large areas of a culture dish, we receive
reliable and well-resolved data on cell areas, with the major-
ity of cells taking up between 150 and 350 mm2 (see Fig. 2).
This distribution corresponds well to previous studies con-
ducted by Puliafito et al. (21).
The determination of Young’s moduli of individual cells
as a function of cell area was performed by AFM indenta-
tion experiments. In these experiments, cell area and
Young’s modulus were extracted concomitantly from the ac-
quired force maps. Therein, cell-cell contacts are marked by
narrow bands of exceptionally stiff behavior, as is confirmed
by overlay with optical phase-contrast images (see Fig. S1).
We decided to use the force data for determination of cell
borders instead of fluorescence images of fixed samples
because cell position and area change dynamically and
could easily vary between the force map acquisition and
successful fixation.
Binning the results of cells in bands of 50 mm2, Fig. 3
shows that for increasing cell area within a monolayer, the
Young’s modulus indeed increases monotonically from
1 kPa to 3 kPa within sizes ranging from about 100 mm2
up to about 600 mm2. Considering that the ratio between
circumference/area decreases for larger areas and because
the circumference usually marks the stiffest region of such
FIGURE 2 Kernel density estimates of projected cell areas obtained from
ZO-1 stainings of confluent MDCK II cells (n ¼ 506). An average cell area
of 278 mm2 is found.
Biophysical Journal 116, 2204–2211, June 4, 2019 2207
Nehls et al.
FIGURE 3 Plot of the cells’ Young’s modulus (median) as a function of
projected apical cell area. Cells within a range of 50 mm2 were collected and
represented in points; error bars represent the standard error of the mean for
all condensed cells’ values. The dotted line (smoothed spline) serves as a
guide to the eye, showing the saturation effect for large areas. Data include
14,326 FDCs taken on 105 cells.
cells, larger areas should systematically decrease in median
stiffness. However, the contrary was observed in our exper-
iments, underlining the actual size-dependent stiffening.
Because the increase in stiffness with occupied area of cells
within a confluent cell monolayer corresponds to our obser-
vation of stiffness changes depending on initial seeding cell
density (Fig. 1), we suggest that the cell area plays a pivotal
role in cell-stiffness homeostasis. Interestingly, the Young’s
modulus increases in a nonlinear fashion and seems to satu-
rate for very large cell sizes.
Single-cell studies
Our experiments concerning size-stiffness correlation of
cells that are part of a confluent layer rely on the natural
size distribution, which is, among other factors, also a result
of seeding density and time. This brings up the question as
to what extent the size dependence of cell stiffness is a
feature of confluent cells and originates from neighboring
cells pulling more strongly to enlarge the area or is, instead,
a generic property. Moreover, the number of cells exhibiting
sizes at the lower or upper end of this distribution is very
low. To force a larger number of cells into extreme sizes
and thereby increase the significance of results, we used mi-
cropatterned culture substrates. These substrates consist of
well-defined areas coated with collagen I to foster adhesion
of cells surrounded by coatings with nonadhesive polymer,
forcing the cells to occupy and adapt their shape to that of
the extracellular matrix patterns. MDCK II cells have shown
that if the patterns are packed densely enough for cells to
form cell-cell contacts and form a confluent layer, the
cell-cell adhesion dominates, and cells essentially disregard
the matrix patterns (unpublished data). Therefore, instances
2208 Biophysical Journal 116, 2204–2211, June 4, 2019
of the patterned grids were separated far enough to allow for
only a single MDCK II cell per instance to adhere, in which
case cells successfully adapt to the matrix pattern.
Single-spread MDCK II cells show a different size distri-
bution compared to confluent ones. They are lower in height
but produce larger adhesion areas. Therefore, we investigated
cells with adhesion areas of 900, 1200, and 1500 mm2. This
approach not only enabled us to enforce a particular cell size
but also an exact adhesion geometry. Using highly symmetric
patterns—here, disks—we can average results by using the
four symmetry axes when analyzing parameter maps. By
doing so and averaging locally over all investigated cells,
we receive parameter maps with high statistical accuracy to
generate a distribution of stiffness values on different areas
of the cell surfaces. See the recent work of Garcia and Garcia
for a detailed discussion on this topic (33).
As shown in Fig. 4, circular cells show a small ring
around their perimeter of high Young’s moduli, surrounding
a larger central region that is rather soft. Although the cen-
tral region with a Young’s modulus of 1.2 kPa is quite
similar to values of other studies on confluent cells, the
stiffer perimeter shows moduli that are up to two orders of
magnitude larger (11,34). The width of this ring differs
slightly depending on the total adhesion area, but the general
pattern is strikingly similar between all three tested sizes.
Notably, the modulus depends also on the distance to under-
lying substrate. The numbers are therefore rather apparent
ones in the context of the corresponding experiment.
The central region on the patterned cells, apart from the
circumference ring, represents the majority of the cell sur-
face. As seen in the one-dimensional kernel density distribu-
tion (Fig. 4, bottom), the Young’s modulus is very similar for
all investigated cell sizes, with virtually no effect from the
FIGURE 4 Elasticity of micropatterned cells. Parameter maps of the log-
arithmic Young’s modulus (top) at a resolution of 1.5  1.5 mm show softer
central regions and stiffer peripheries. For all adhesion areas (blue ¼
900 mm2, red ¼ 1200 mm2, green ¼ 1500 mm2), the Young’s modulus of
the central region is almost the same at 1.2 kPa. The stiffer perimeter has
no dedicated peak in the one-dimensional distribution, with values up to
two orders of magnitude larger than the softer interior part. Nblue ¼ 2834,
Nred ¼ 2924, Ngreen ¼ 2362. To see this figure in color, go online.

different adhesion areas. The stiffer perimeter of the cells,
however, displays a size-dependent Young’s modulus, i.e.,
E900 ¼ 73.0 kPa, E1200 ¼ 18.6 kPa, and E1500 ¼ 29.9 kPa.
In contrast to the confluent studies, this is not a simple stiff-
ening with increased size because the intermediate adhesion
area results in the softest cell periphery, whereas the small-
est adhesion area leads to the softest cell periphery. These
results suggest that the mechanical behavior of MDCK II
cells follow distinctively different rules depending on
whether or not a confluent layer is present, which further
supports the idea of cell mechanics playing a role in layer
organization and vice versa. Still, these results are surpris-
ing, given recent studies by Efremov et al. who compared
the stiffness of vero cells either in single-spread or confluent
stages and observed stiffer behavior for single cells in a
setup similar to ours (35). Also, similar studies of single
cells on adhesive islands (human mesenchymal stem cells
and lung human microvascular endothelial cells) showed
size stiffening in the respective studies, which we did not
observe in our experiments on MDCK II cells (36,37).
DISCUSSION
The mechanical properties of epithelial cells are of major
importance for their biological function as efficient barrier
tissue. Therefore, epithelia have to ensure that their struc-
tural integrity is not compromised. One prime example for
such cells is the MDCK II cell line, which forms a rather
flexible and fluid two-dimensional layer of cells where inter-
cellular gaps are tightly closed to provide control over the
transepithelial flow. In this study, we used MDCK II cells
grown on standard culture surfaces and performed site-spe-
cific nanoindentation experiments to probe the repulsive
force in response to deformation. This permitted us to
gain information about the apical mechanics of these cells.
We employ simple Hertzian mechanics here and refrain
from more complex viscoelastic models to keep the matter
simple. Essentially, more sophisticated models based on
shell mechanics also generate the same qualitative picture.
The goal of this work was to elaborate on the distribution
of mechanical properties between multiple cells within
confluent layers and to search for correlations between
cellular mechanics and the occupied area of cells within
these layers. From a tensegrity point of view, we expect
larger structures if neighboring cells exert stress through
adherens junctions, leading to larger restoring forces upon
indentation.
Therefore, we initially performed rheological experiments
on samples derived from cells seeded at different initial cell
densities. Force indentation curves were subjected to a
theoretical framework that has been well established
before (9), and we found Young’s moduli that are comparable
to those found in previous studies (11,15,38). However,
by comparing the results of samples with different cell den-
sities, we found that there is a significant trend to decreasing
Cell Size Mechanics
stiffness for higher amounts of cells seeded. Higher amounts
of seeded cells do also result in higher cell density after an
equal amount of incubation time and therefore lead to smaller
cell areas (see Fig. S4). Apart from the decreasing Young’s
modulus, we also found that the loss tangent at 100 Hz drops
moderately with decreasing cell-seeding density. This means
that cells behave more like solids if larger. This corresponds
well to the general behavior of cells in the framework of soft
glassy materials in which stiffer cells also generate less en-
ergy dissipation. However, care needs to be taken when cell
densities are very low because polarization of cells might
not be completed yet (39). These findings once more under-
line the importance of using equal incubation conditions for
all samples to provide correct comparability between
measurements.
Further, given the correlation between seeding and incu-
bation conditions with both elasticity and cell adhesion area,
we wanted to elucidate whether we can also find a correla-
tion between cell size and elasticity in a single confluent
layer independent of seeding density or other factors. Fortu-
nately, even monoclonal cultures show cells with a broad
size distribution, and although the exact regulatory mecha-
nisms involved in size homeostasis are still under debate, in-
fluence of cell size on processes like division rates has been
shown to exist (24,40,41). Alterations of cell mechanics
depending on size may pose a way for cells to recognize
their current size and alter their biochemistry appropriately.
This is especially interesting because different stiffness and
thereby actomyosin network architectures can carry infor-
mation even from mother to daughter cells and should there-
fore contribute to the ongoing discussion on how cells could
be aware of their very own size (42).
We have shown that for MDCK II cells, a decently broad
size distribution can be obtained, and by determining the
exact size and median Young’s modulus of individual cells,
we were able to show that larger cells tend to have higher
stiffness even if originating from the same cell layer. Inter-
estingly, we found a trend including saturation effects for
large sizes. Such saturation has also been observed when
investigating cells seeded on gels of different elasticity,
where cell stiffness correlates with substrate stiffness (37).
Interestingly, we did not find a window of optimal size
compared to Miettinen et al., who observed a peak metabolic
activity for an optimal cell size (43). This shows that the way
cellular properties correlate with cell size is diverse and com-
plex in nature, and cell stiffness is one of these properties.
Lastly, we tested whether or not we can use single
patterned cells to find the same correlations we found in
the confluent samples already. In other words, we wanted
to find out whether the size-dependent stiffness is a univer-
sal feature or only occurs in confluent layers. Although we
had to rely on much larger adhesion areas for single-cell
studies, this approach provided better reproducibility of
exact cell sizes and yielded laterally resolved parameter
maps of the apical mechanics of cells (44). The Young’s
Biophysical Journal 116, 2204–2211, June 4, 2019 2209
Nehls et al.
moduli of the stiffer, peripheral region of single patterned
MDCK II cells do depend on the total adhesion area,
whereas the central region shows similar Young’s moduli
regardless of the adhesion area. Previous studies by Tee
et al. and Roca-Cusachs et al. did not consider this differ-
ence in behavior depending on the exact location on the
cell surface and could not report such bimodal distribution
because the authors did not acquire force maps across the
complete cell surface. It is therefore even more surprising
that neither of the surface regions, which we managed to
distinguish by mechanical behavior, show the same changes
that have been observed on other cell lines in the respective
studies. This emphasizes how fundamentally different the
mechanical behavior of cells is regulated depending on
cell type and how strongly it is influenced by the environ-
mental conditions.
In larger cells, contractile forces and internal stresses are
presumably enhanced. A similar trend was previously found
when cells were cultured on pores. Cells on larger pores
were smaller and softer (32).
CONCLUSION
We scrutinized the connection between the size of cells and
their apical mechanics, showing that in confluent MDCK II
cells, stiffness is increased in samples with lower initial cell-
seeding density. Even in one cell monolayer, the stiffness of
individual cells is increased with increasing apical surface
area. This shows that special caution is required when
comparing mechanical studies of cells carried out under
different culture conditions.
Interestingly, this behavior could not be observed for sin-
gle patterned cells, where we found that mechanics differ be-
tween certain locations on a cell’s surface, but the stiffness
obtained from the cell’s center does not depend on the cell’s
size. This emphasizes the importance of cell-cell contacts for
the elastic response of cells, as recently shown (38). Taken
together, these findings show that cellular mechanics react
to environmental conditions in complex ways and that ten-
sion homeostasis and cell size are inherently coupled. The
further understanding of the correlation between mechanical
and biochemical properties of cells should ultimately lead to
a better understanding of how these cells are able to respond
appropriately to a large variety of different environmental
conditions and still maintain performance.
SUPPORTING MATERIAL
Supporting Material can be found online at https://doi.org/10.1016/j.bpj.
2019.04.028.
AUTHOR CONTRIBUTIONS
H.N. and F.R. performed experiments and analysis of the experiments
regarding different cell-seeding densities. S.N., H.N., and S.K. performed
2210 Biophysical Journal 116, 2204–2211, June 4, 2019
experiments on confluent cells related to individual cell size. S.N. per-
formed analysis on these data sets. Single-cell studies were performed
and analyzed by S.N. S.N. and A.J. wrote the manuscript.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from SFB 937 (A17) and SPP
1782. H.N. additionally thanks the Deutsche Telekom Stiftung for financial
support.
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Biophysical Journal, Volume 116

Supplemental Information

Stiffness of MDCK II Cells Depends on Confluency and Cell Size

Stefan Nehls, Helen Nöding, Susanne Karsch, Franziska Ries, and Andreas Janshoff
 Supporting Material

Stiffness of MDCK II Cells Depends on Confluency and

Cell Size
S. Nehlsa , H. Nödinga , S. Karscha , F. Riesa , A. Janshoffa∗
a
Georg-August-Universität Göttingen, Institute of Physical Chemistry,
37077 Göttingen, Germany

Figure S1: Stiffness map showing the slope of a linear fit of the last 20 nm of each FDC (top left, green).
Cell-cell borders are marked by bright areas. Comparing this to the phase-contrast image of the same
sample, nuclei and cell borders are visible (bottom left). The right image shows an overlap of both
images, confirming that cell-cell contacts are identified in stiffness maps. Small deviations between both
images can occur due to thermal drift since the acquisition of the force map takes several minutes. Box
sizes are 100 µm x 100 µm.

1
Figure S2: Representative height topography scan and associated force map analyzed with the Hertzian
contact model implemented in the JPK Data Processing Software (quadratic pyramid tip shape with half-
angle to edge of 17.5◦ and ν = 0.5) showing elevated Young’s moduli at cell-cell junctions.

2
Figure S3: Proof-of-principle of the fit routine to obtain the Young’s modulus from force distance curves.
Force distance data is simulated for a sample stiffness of 1 kPa and an indentation up to a force of 500 pN
(top, blue). The data was realistically sampled and Gaussian noise was added with typical amplitude.
The resulting fit is shown as dashed red line. The process was repeated for 10,000 times (center), each
time with random noise, and the distribution of the received modules from the noisy data are shown as
a kernel density plot (blue), with the red line marking the originally simulated Young’s modulus. The
real modulus is very well represented by the results. Fits can be filtered by the fit quality (bottom panel),
where interestingly higher quality factor, hence larger deviations of the fit, lead to a higher number of fits
that are accepted and a better overall estimation of the real modulus. This also shows that for our data,
quality factors larger than one can be just due to the random noise.

3
A1 A2 A3 A4

270 c/mm²

B1 B2 B3 B4 40 µm

540 c/mm²

Figure S4: Examplary immunostainings of MDCK II cells at either 270 c/mm2 (A) or 540 c/mm2 (B).
Shown are the basal (1) and apical (2) layers of f-actin stainings and E-cadherin (3) and ZO-1 (4) stainings.
All show a densely packed and closed cell monolayer despite large cell size differences.