Outline of Proposed Research Page 1 of 1 Wontae Lee (PIN 484685)

Background. Cell-generated forces play a critical role in biological process including development,
wound healing, and disease progression. Human physiology is in 3D, but the vast majority of current cell
force quantification approaches are limited to 2D tissue analyses. While some 3D alternatives exist, 3D
traction force microscopy cannot be performed in vivo1, and imaging the deformation of incompressible
oil microdroplets can only measure local differences around the microdroplet and not absolute cell
forces2. Moreover, the complex biomechanics created by the interaction of cells and the extracellular
matrix require monitoring over time, and the anisotropy and heterogeneity of real tissues require
localised measurements of internal tissue structures. Biology occurs dynamically as cells change with
disease and age, and measuring the real-time changes in cell force is crucial to further our understanding
of mechanobiology. Hence, there is a critical need to engineer a novel mechanosensor that can
measure local, absolute cell-generated forces in 3D tissues, with real-time, in vivo capabilities.

Rationale. We hypothesise that measuring the deformation in soft, compressible mechanosensors

embedded within tissue can be used to quantify changes in cell-generated mechanical forces (Fig.
1). The specific objectives of this research are to: (1) fabricate and validate the mechanosensor
technology in well-controlled samples; (2) investigate mechanically dynamic systems to quantify rapidly
changing force profiles; and (3) investigate their application as surrogate read-outs in drug discovery by
measuring functional tissue activity. Mechanosensor deformations will be monitored by in situ imaging
techniques, which is advantageous as the functional activity read-out provided by imaging is non-
invasive and low cost compared to staining and other specialized molecular biology approaches.

Fig. 1 Cell-generated forces can

be quantified based on the
measured deformations of a soft,
compressible mechanosensor.

Methods. Soft, compressible, and biologically compatible BaSO4-polyacrylamide3 (PA) hydrogel

microspheres (mechanosensor) will be fabricated by dispersing picolitre volumes of PA prepolymer in
an immiscible oil bath via mechanical agitation. BaSO4 is a radiocontrast agent visible under X-ray
radiation, and will enable imaging at micron-scale resolution into deep tissue. The elastic modulus of the
material will be evaluated using bulk shear rheology (~0.2 kPa), and sizes determined using micro-CT
(~50 µm). A finite element model will be developed to extract tissue forces based on measured microgel
deformations and elastic modulus. Microgels will be embedded into PA matrices undergoing stretch and
collagen contractile tissues to measure force during remodelling (Aim 1). Microgels will be embedded
within engineered cardiomyocyte spheroids to measure force of beating with electrical stimulation (Aim
2), and force generation as a result of applied drugs (Aim 3).

Significance. The mechanosensor proposed in this study will enable the real-time measure of absolute
cell forces in 3D tissues. The incorporation of a radiocontrast agent will enable detection under X-ray
imaging, and may thus ultimately find application in clinical settings. For example, they may provide a
non-invasive alternative to the endomyocardial biopsy in providing routine organ rejection surveillance.
Broadly applicable, the mechanosensor may be translated to other studies in the sciences and engineering
for the functional, real-time measurement of cell forces. For example, they may be leverage to develop
physiologically realistic organ-on-a-chip devices to screen novel drug compounds, study disease
etiology, and investigate the mechanics driving developmental biology.

1. Legant, W. R. et al. (2010) Nature Methods. 7(12): 969–971.

2. Campàs, O. et al. (2014) Nature Methods. 11(2): 183–189.
3. Wang Q. et al. (2013) Green Chemistry. 15: 2222–2229.