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The cellular microenvironment has been shown to play a fundamental role in the regulation of cell function, stem cell fate determination, maintenance of cell potency and tissue homeostasis. Our laboratory focuses on the study of the effects of cellular microenvironment in the context of cancer and neurological models, based on the observation that a healthy environment can induce the suppression of tumorigenesis in mouse models. Insights concerning the molecular mechanisms that drive these processes are very limited, partly due to the inability of the current traditional methods of investigation, such as two-dimensional cell cultures and animal models, to accurately represent the human in vivo cellular microenvironment. Three-dimensional cell cultures allow to overcome the structural limitations posed by monolayer cultures, and maintain the ease of experiment design, monitoring and data analysis associated with in vitro procedures. Our laboratory has established systems to overcome some of these limitations and rely on the strengths of three-dimensional culture methods to elucidate mechanisms that govern stem cell differentiation. A customized 3D extrusion-based bioprinter was developed starting from a commercially available model, allowing for precise and controlled injection of cells within three-dimensional substrates. This tool allows for design of highly controlled experiments, in which the effects of cellular microenvironment on stem cell differentiation can be studied at a single-cell resolution. For increased levels of biomimicry, tissue specific substrates are generated from extracted tissue. Collected tissue is subjected to a chemical decellularization process, followed by lyophilization, enzymatic digestion and neutralization, to generate a self-gelling product upon incubation at 37°C. Mammary and brain extracellularmatrix-derived substrates have been shown to support the growth of cells of the epithelial and neuronal lineages, respectively. Here, we apply these established systems to study the effects of the environment constituted by the three-dimensional substrates on the differentiation of injected stem cells. |
