Abstract
Fluid flow in biological tissues is important in both mechanical and biological contexts. Given the hierarchical nature of tissues, there are varying length-scales at which timedependent mechanical behavior due to fluid flow may be exhibited. Here, spherical nanoindentation and microindentation testing are employed for the characterization of length-scale effects in the mechanical response of hydrated tissues. This is a fast and robust technique for characterizing the hierarchical structure of biological materials from nanometer to micrometer length-scales, including making quantitative material property measurements to identify length-scale effects that are cell-relevant. During the course of embryological development, stem cell differentiation and morphogenesis occur as a result of dynamic changes in the molecular composition and structure of the extracellular microenvironment. Thus, the development of approaches to systematically control biochemical and biophysical cues comprising the microenvironment of pluripotent and multipotent cells could facilitate an improved understanding of fundamental principles and mechanisms of stem cell and developmental biology. With this goal in mind, we have focused on spatially and temporally engineering the microenvironments of 3D stem cell aggregates to create robust and reproducible systems to investigate the dynamics of differentiation and morphogenesis in vitro. For example, macroscopic hydrodynamic forces (imparted by rotary orbital suspension culture) enhance the efficiency, yield and homogeneity of pluripotent embryonic stem cell (ESC) spheroids referred toas “embryoid bodies” (EB). Hydrodynamic conditions impact global transcriptional activity and subsequent differentiation of EB cell populations to different germ lineages via modulation of intracellular signalling pathways, such as ß-catenin. In order to locally control the biochemical milieu of the microenvironment within 3D multicellular aggregates, microparticles of different materials were physically entrapped in stem cell spheroids without adversely affecting cell viability or intercellular adhesion. Presentation of morphogenic factors from microparticles of different materials induced gross morphological and phenotypic differences in the spatial and temporal patterning of ESC fates compared to soluble delivery methods. Incorporation of paramagnetic microparticles within cell aggregates enabled non-contact manipulation of spheroid populations via magnetic forces and the spatial patterning of complex multicellular structures. Altogether, these results demonstrate that regulating stem cell 3D environments via the combination of macro- and microscale enabling technologies can yield more effective and efficient strategies to direct differentiation and morphogenesis of stem cells. In addition to yielding new insights into stem cell and developmental biology, these engineering based strategies offer novel routes for stem cell-based tissue engineering and biomanufacturing.