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.

A pH-sensor scaffold for mapping spatiotemporal gradients in three-dimensional in vitro tumour models.

The detection of extracellular pH at single cell resolution is challenging and requires advanced sensibility. Sensing pH at high spatial and temporal resolution might provide crucial information in understanding the role of pH and its fluctuations in a wide range of physio-pathological cellular processes, including cancer. Here, a method to embed silica-based fluorescent pH sensors into alginate-based three-dimensional (3D) microgels tumour models, coupled with a computational method for fine data analysis, is presented. By means of confocal laser scanning microscopy, live-cell time-lapse imaging of 3D alginate microgels was performed and the extracellular pH metabolic variations were monitored in both in vitro 3D mono- and 3D co-cultures of tumour and stromal pancreatic cells. The results show that the extracellular pH is cell line-specific and time-dependent. Moreover, differences in pH were also detected between 3D monocultures versus 3D co-cultures, thus suggesting the existence of a metabolic crosstalk between tumour and stromal cells. In conclusion, the system has the potential to image multiple live cell types in a 3D environment and to decipher in real-time their pH metabolic interplay under controlled experimental conditions, thus being also a suitable platform for drug screening and personalized medicine.

Co-loading of doxorubicin and iron oxide nanocubes in polycaprolactone fibers for combining Magneto-Thermal and chemotherapeutic effects on cancer cells.

Among the strategies to fight cancer, multi-therapeutic approaches are considered as a wise choice to put in place multiple weapons to suppress tumors. In this work, to combine chemotherapeutic effects to magnetic hyperthermia when using biocompatible scaffolds, we have established an electrospinning method to produce nanofibers of polycaprolactone loaded with magnetic nanoparticles as heat mediators to be selectively activated under alternating magnetic field and doxorubicin as a chemotherapeutic drug. Production of the fibers was investigated with iron oxide nanoparticles of peculiar cubic shape (at 15 and 23 nm in cube edges) as they provide benchmark heat performance under clinical magnetic hyperthermia conditions. With 23 nm nanocubes when included into the fibers, an arrangement in chains was obtained. This linear configuration of magnetic nanoparticles resemble that of the magnetosomes, produced by magnetotactic bacteria, and our magnetic fibers exhibited remarkable heating effects as the magnetosomes. Magnetic fiber scaffolds showed excellent biocompatibility on fibroblast cells when missing the chemotherapeutic agent and when not exposed to magnetic hyperthermia as shown by viability assays. On the contrary, the fibers containing both magnetic nanocubes and doxorubicin showed significant cytotoxic effects on cervical cancer cells following the exposure to magnetic hyperthermia. Notably, these tests were conducted at magnetic hyperthermia field conditions of clinical use. As here shown, on the doxorubicin sensitive cervical cancer cells, the combination of heat damage by magnetic hyperthermia with enhanced diffusion of doxorubicin at therapeutic temperature are responsible for a more effective oncotherapy.