A quantitative approach for determining the role of geometrical constraints when shaping mesenchymal condensations

In embryogenesis, mesenchymal condensation is a critical event during the formation of many organ systems, including cartilage and bone. During organ formation, mesenchymal cells aggregate and undergo compaction while activating developmental programmes. The final three-dimensional form of the organ, as well as cell fates, can be influenced by the size and shape of the forming condensation. This process is hypothesized to result from multiscale cell interactions within mesenchymal microenvironments; however, these are complex to investigate in vivo. Three-dimensional in vitro models that recapitulate key phenotypes can contribute to our understanding of the microenvironment interactions regulating this fundamental developmental process. Here we devise such models by using image analysis to guide the design of polydimethylsiloxane 3D microstructures as cell culture substrates. These microstructures establish geometrically constrained micromass cultures of mouse embryonic skeletal progenitor cells which influence the development of condensations. We first identify key phenotypes differentiating face and limb bud micromass cultures by linear discriminant analysis of the shape descriptors for condensation morphology, which are used to guide the rational design of a micropatterned polydimethylsiloxane substrate. High-content imaging analysis highlights that the geometry of the microenvironment affects the establishment and growth of condensations. Further, cells commit to establish condensations within the first 5 h; condensations reach their full size within 17 h; following which they increase cell density while maintaining size for at least 7 days. These findings elucidate the value of our model in dissecting key aspects of mesenchymal condensation development.