Historically, the cell culture experiments underlying biological understanding and drug candidate screening
have been performed with homogeneous populations of cells on flat, stiff plastic substrates. Mounting evidence
suggests that these stiff, static culture substrates only widen the gap between in vitro and in vivo assays. For
many stem cells and multicellular constructs, 2D substrates are unable to recapitulate the tissue architecture
and elicit biologically relevant responses. The extracellular matrix (ECM) surrounding cells in vivo is a complex,
heterogeneous, and dynamic environment that exchanges biophysical and biochemical cues with cells. ECM
mechanics and composition change as a function of time—during development, regeneration, repair, disease
progression, and aging—and as a function of 3-dimensional location in organs. To increase the physiological
relevance of in vitro cell culture experiments, researchers have developed biological and synthetic matrices
that approximate the mechanics and water content of tissue. These ECM mimics represent a compromise
between complexity and control. We will address two key challenges for 4D cell culture: reversible external
control over the matrix, and independently tunable stiffness and stress relaxation.
The objective of the proposed research is to develop OptoGels, a tunable platform for 4D cell culture
technology. We have developed viscoelastic hydrogels that can be stiffened and softened by two different
wavelengths of visible light. By installing a reversible photoswitch, azobenzene, adjacent to a dynamic covalent
boronic ester linkage, we can control the binding constant for boronic ester formation with light. In preliminary
experiments, we have achieved phototunable stiffnesses up to 9 kPa, spatiotemporal patterning of stiffness,
and cytocompatibility. The Specific Aims of the proposed research are to (1) establish design parameters for
OptoGels to maximize their versatility as cell culture materials, (2) benchmark mesenchymal stem cell and
epithelial cell mechanobiology in OptoGels against literature data and commercial materials, and (3) engineer
spatially controlled OptoGels that enhance the functional maturation of pluripotent stem cell-derived
hepatocytes. Functional readouts and metrics that can be compared to established materials include YAP
nuclear translocation, stem cell differentiation, cell spreading, cell migration, gene expression, and enzyme
activity. The anticipated products of this research are a suite of OptoGel formulations with mechanical
properties that can be tuned over physiologically relevant length and time scales. The long-term goal of this
research is to develop user-defined cell culture materials that allow biomedical researchers to understand and
control cellular processes relevant to human health.