REVIEW
Tissue Cells Feel and Respond to the
Stiffness of Their Substrate
Dennis E. Discher,
1
*
Paul Janmey,
1
Yu-li Wang
2
Normal tissue cells are generally not viable when suspended in a fluid and are
therefore said to be anchorage dependent. Such cells must adhere to a solid, but a
solid can be as rigid as glass or softer than a baby’s skin. The behavior of some cells
on soft materials is characteristic of important phenotypes; for example, cell growth
on soft agar gels is used to identify cancer cells. However, an understanding of how
tissue cells—including fibroblasts, myocytes, neurons, and other cell types—sense
matrix stiffness is just emerging with quantitative studies of cells adhering to gels
(or to other cells) with which elasticity can be tuned to approximate that of tissues.
Key roles in molecular pathways are played by adhesion complexes and the actin-
myosin cytoskeleton, whose contractile forces are transmitted through transcellular
structures. The feedback of local matrix stiffness on cell state likely has important
implications for development, differentiation, disease, and regeneration.
Anchorage dependence refers to a cell_s need
for adhesion to a solid. Most tissue cells are
simply not viable upon dissociation and sus-
pension in a fluid, even if soluble proteins are
added to engage cell adhesion molecules Ee.g.,
integrin-binding RGD peptide (1, 2)^.Fluids
are clearly distinct from solids in that fluids
will flow when stressed, whereas solids have
the ability to resist sustained pushing and pull-
ing. In most soft tissues—skin, muscle, brain,
etc.—adherent cells plus extracellular matrix
contribute together to establish a relatively
elastic microenvironment. At the macro scale,
elasticity is evident in a solid tissue_s ability to
recover its shape within seconds after mild pok-
ing and pinching, or even after sustained com-
pression, such as sitting.
At the cellular scale, normal tissue cells
probe elasticity as they anchor and pull on
their surroundings. Such processes are de-
pendent in part on myosin-based contractility
and transcellular adhesions—centered on in-
tegrins, cadherins, and perhaps other adhesion
molecules—to transmit forces to substrates. A
normal tissue cell not only applies forces but
also, as reviewed here, responds through
cytoskeleton organization (and other cellular
processes) to the resistance that the cell senses,
regardless of whether the resistance derives
from normal tissue matrix, synthetic substrate,
or even an adjacent cell. Furthermore, physical
properties of tissues can change in disease Eas
imaged now by magnetic resonance imaging
(MRI) or ultrasound elastography (3–5)^,and
cellular responsiveness to matrix solidity can
likewise change, as illustrated by the growth of
cancer cells on soft agar Ee.g., (6)^.
Contractile forces in cells are generated by
cross-bridging interactions of actin and myosin
filaments. For adherent cells, some of these
forces are transmitted to the substrate (referred
to as traction forces) and cause wrinkles or
strains when the substrate consists of a thin
film or a soft gel (7–12) (Fig. 1A). The cell, in
turn, is shown to respond to the resistance of
the substrate, by adjusting its adhesions, cyto-
skeleton, and overall state. Although con-
siderable attention has been directed at the
responsiveness of individual cells to external
forces (outsideYin) that range from fluid flow
to direct stretching and local twisting (13), we
are now beginning to understand that cellular
responses to cell-exerted forces involve a
feedback loop of insideYoutsideYin that
couples to the elasticity of the extracellular
microenvironment. An analogy to muscle
building is perhaps useful: A bicep is not built
by passive flexing; the muscle must do active
work against a load. Moreover, a load of 1 kg
clearly feels different from a load of 2 kg.
Similar sensitivity, growth, and remodeling
principles seem to apply to most anchored
cells.
On ligand-coated gels of varied stiffness,
epithelial cells and fibroblasts (14)werethe
first cells reported to detect and respond dis-
1
School of Engineering and Applied Science and Cell
and Molecular Biology Graduate Group, University of
Pennsylvania, Philadelphia, PA 19104–6315, USA.
2
Departments of Physiology and Cell Biology, Uni-
versity of Massachusetts, Worcester, MA 01655, USA.
*To whom correspondence should be addressed.
E-mail: discher@seas.upenn.edu
Fig. 1. Substrate strain and tissue stiffness. (A) Strain distribution computed in a soft matrix
beneath a cell. The circular cell has a uniform and sustained contractile prestress from the edge to
near the nucleus (81). (B) Stress versus strain illustrated for several soft tissues extended by a force
(per cross-sectional area). The range of slopes for these soft tissues subjected to a small strain gives the
range of Young’s elastic modulus, E, for each tissue (24, 28, 30). Measurements are typically made on
time scales of seconds to minutes and are in SI units of Pascal (Pa). The dashed lines (- - -) are those for
(i) PLA, a common tissue-engineering polymer (89); (ii) artery-derived acellularized matrix (90); and (iii)
matrigel (42).
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tinctly to soft versus stiff substrates. Although
molecular pathways are still only partially
known, muscle cells, neurons, and many other
tissue cells have since been shown to sense
substrate stiffness (15–17). Unlike cells on soft
gels or in tissues, cells cultured on tissue-
culture plastic or glass coverslips are attached
(often via adsorbed matrix protein) to essen-
tially rigid materials. The question therefore
arises: Do cells perceive and respond to the
rigidity of these conventional materials in
ways that contrast with their behavior in
much more compliant tissues, gels, or sub-
layers of cells? The increasingly clear, af-
firmative answer to this question appears
important in its impact not just on standard
cell culture but also, perhaps, in understand-
ing disease processes, morphogenesis, and
tissue-repair strategies.
Soft Tissue Benchmarks
Cells adhere to solid substrates that range in
stiffness from soft to rigid and that also vary in
topography and thickness (e.g., basement mem-
brane). Regardless of geometry, the intrinsic
resistance of a solid to a stress is measured by
the solid’s elastic modulus E, which is most
simply obtained by applying a force—such
as hanging a weight—to a section of tissue
or other material and then measuring the
relative change in length or strain (Fig. 1B, inset).
Another common method to obtain E involves
controlled poking by macro- and micro-indenters,
including atomic force microscopes (AFMs)
(18, 19). Many tissues and biomaterials exhibit a
relatively linear stress versus strain relation up to
small strains of about 10 to 20%. The slope E of
stress versus strain is relatively constant at the
small strains exerted by cells (20), although
stiffening (increased E) at higher strains is the
norm (21, 22). Nonetheless, microscopic views
of both natural and synthetic matrices [e.g.,
collagen fibrils and polymer-based mimetics
(23)] suggest that there are many subtleties to
tissue mechanics, particularly concerning the
length and time scales of greatest relevance to
cell sensing. Sample preparation or state is
another obvious issue; for example, elastic
moduli of whole brain in macroscopic mea-
surements can vary by a factor of 2 or more,
depending on specifics of preparation, tissue
perfusion, etc. (24). In addition, with cells as
well as tissues, many probing methods involve
high-frequency stressing (25), whereas relevant
time scales for cell-exerted strains seem likely
to range from seconds to hours, motivating long
time studies of cell rheology [recent cell me-
chanics references (26, 27)]. Regardless, com-
parisons of three diverse tissues that contain a
number of different cell types show that brain
tissue is softer than muscle (28, 29), and muscle
is softer than skin (30) (Fig. 1B). Although
mapping soft tissue micro-elasticities at a
resolution typical in histology seems impor-
tant, the implication here is that there are dis-
tinct elastic microenvironments for epithelial
cells and fibroblasts in skin, for myotubes in
fiber bundles, and for neurons in brain.
Correlations have long been made between
increased cell adhesion and increased cell
contractility [e.g., (31)], but it now seems
clear that tactile sensing of substrate stiffness
feeds back on adhesion and cytoskeleton, as
well as on net contractile forces, for many cell
types. Seminal studies on epithelial cells and
fibroblasts exploited inert polyacrylamide gels
with a thin coating of covalently attached
collagen (14). This adhesive ligand allows the
cells to attach and—by controlling the extent
of polymer cross-linking in the gels—E can
be adjusted over several orders of magnitude,
from extremely soft to stiff. Images of adhe-
sion proteins such as vinculin are revealing
(Fig. 2, top): Soft, lightly cross-linked gels
(EÈ 1 kPa) show diffuse and dynamic adhesion
complexes. In contrast, stiff, highly cross-
linked gels (E È 30 to 100 kPa) show cells
with stable focal adhesions, typical of those
seen in cells attached to
glass. Similarly, rigidifi-
cation of cell-derived
three-dimensional (3D)
matrices alters 3D-matrix
adhesions, because the
adhesions are replaced
by large, nonfibrillar fo-
cal adhesions similar to
those found on fixed 2D
substrates of fibronectin
(32). Consistent with a
role for signaling in stiff-
ness sensing, tyrosine
phosphorylation on mul-
tiple proteins (including
paxillin) appears broad-
ly enhanced in cells on
stiffer gel substrates (14),
whereas pharmacologi-
cally induced, nonspecif-
ic hyperphosphorylation
drives focal adhesion for-
mation on soft materials.
Inhibition of actomyosin
contractions, in contrast,
largely eliminates promi-
nent focal adhesions,
whereas stimulation of
contractility drives in-
tegrin aggregation into
adhesions (33). Ad-
ditionally, although mi-
crotubules have been
proposed to act as ‘‘struts’’
in cells and thus limit
wrinkling of thin films
by cells (34), quantifica-
tion of their contribu-
tions to cells on gels
shows that they provide
only a minor fraction of
the resistance (14%) to contractile tensions;
most of a cell’s tension is thus resisted by
matrix (35).
Traction stresses (t, force per area) exerted
by fibroblasts on gels were the first to be
mapped by embedding fluorescent microbeads
near the gel surface and then imaging bead
displacements before and after cell detachment
(10, 20). Although larger tractions are exerted
on stiffer gels, typical tractions of btÀÈ1kPa
exceed by orders of magnitude the viscous
fluid drag on any cell crawling in culture. In
addition, mean cell tractions equate to mean
gel strains that differ very little (e
out
0 bt/EÀ;
3 to 4%) between gels that differ by twofold
in E.Thissuggeststhate
out
is sensed by cells
as a tactile set-point, perhaps analogous to
other physiological set-points such as extra-
cellular ion concentrations or optimal growth
factor concentrations. Furthermore, if matrix
strain is approximately constant, then cells
on soft gels need be less contractile than on
stiff gels, and if they are less contractile, then
Fig. 2. Substrate stiffness influences adhesion structures and dy-
namics (14), cytoskeleton assembly and cell spreading (17, 42), and
differentiation processes such as striation of myotubes (28). (Top) The
arrows point to dynamic adhesions on soft gels and static, focal
adhesions on stiff gels. [Adapted from (14)] (Middle) The actin cyto-
skeleton. (Bottom) A cell-on-cell layering in which the lower layer is
attached first to glass so that the upper layer, which fuses from
myoblasts that are added later, perceives a soft, cellular substrate.
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