INVESTIGATING MAMMALIAN CELL NANOMECHANICS WITH SIMULTANEOUS OPTICAL AND ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 384

during migration and other physiological processes. 90-99 In early studies,
cells were grown on silicone gels where gel wrinkling corresponds to the
magnitude of cellular traction forces. 90-92 To quantify traction forces, cells
are often grown on soft deformable substrates which are embedded with
iduciary luorescent tracking particles. 94,99 In many TFM applications,
bead displacements are measured during cell migration. As the material
properties of the deformable substrate are known and controllable, these
bead displacements can be converted into forces, allowing local maps
of traction force to be created. 94,99 Several important early studies have
demonstrated the usefulness and biological relevance of TFM in the study of
cellular nanomechanics. 90-101 Typically, substrates of polyacrylamide, gelatin
(GE) or polydimethylsiloxane pillars have been used successfully and have
revealed striking examples of how living cells respond and affect their local
mechanical environments.

Here, we present a method in which a biocompatible glutaraldehyde
cross-linked GE (GXG) substrate, with 200 nm luorescent beads, can be
poured directly into a standard tissue culture dish (or onto any other
substrate) in a simple one-step approach ( Fig. 18.10 ). The GXG substrate
has a high melting point (>60 ° C) allowing for mammalian cell culture, it
is completely biocompatible without further surface functionalization (but
able to be functionalized if necessary), it is optically clear allowing for
luorescence microscopy and the substrate stiffness can be controlled by
varying the percentage of GE. Finally, we demonstrate the application of
simultaneous traction and atomic force microscopy (TAFM).
Biocompatible GXG gels for TAFM were produced from 5% solutions of
GE. 200 nm red or green luorescent microspheres were mixed thoroughly
with the GE solution. Then the GE was cross-linked with glutaraldehyde
and spread evenly over the surface of a 60 mm plastic culture dish. No
functionalization of the surface was required for cell growth but typical
surface molecules (poly-L-lysine, FN, gelatine) were found to be compatible
with the GXG substrate ( Fig. 18.10 ). GXG substrates were found to have a
Young's modulus of ~28 kPa. C2C12 muscle myoblast cells were used as they
are inherently sensitive to mechanical force. Mechano-stimulation of these
cells is a critical step in the myogeneic pathway during muscle formation
that involves the ability of these cells to apply and generate traction forces
within their micro-environment. Therefore, we expect them to respond
and alter their cellular traction force dynamics in response to mechanical
stimulation with the AFM.

94-96,99,102